Abstract: During secondary bone healing, different tissue types are formed within the fracture callus depending on the local mechanical and biological environment. Our aim was to understand the temporal succession of these tissue patterns for a normal bone healing progression by means of a basic mechanobiological model. The experimental data stemmed from an extensive, previously published animal experiment on sheep with a 3 mm tibial osteotomy. Using recent experimental data, the development of the hard callus was modelled as a porous material with increasing stiffness and decreasing porosity. A basic phenomenological model was employed with a small number of simulation parameters, which allowed comprehensive parameter studies. The model distinguished between the formation of new bone via endochondral and intramembranous ossification. To evaluate the outcome of the computer simulations, the tissue images of the simulations were compared with experimentally derived tissue images for a normal healing progression in sheep. Parameter studies of the threshold values for the regulation of tissue formation were performed, and the source of the biological stimulation (comprising e.g. stem cells) was varied. It was found that the formation of the hard callus could be reproduced in silico for a wide range of threshold values. However, the bridging of the fracture gap by cartilage on the periosteal side was observed only (i) for a rather specific choice of the threshold values for tissue differentiation and (ii) when assuming a strong source of biological stimulation at the periosteum.
Abstract: BACKGROUND: Mesenchymal stem cell (MSC) therapy has the potential to enhance muscular regeneration. In previous publications, our group was able to show a dose-response relationship in female animals between the amount of transplanted cells and muscle force. The impact of sex on the regeneration of musculoskeletal injuries following MSC transplantation remains unclear. OBJECTIVE: We investigated histologic and biomechanical regeneration parameters in rats after autologous transplantation of MSCs. Our hypothesis was that female rats have greater muscle regeneration potential than male rats after autologous MSC transplantation. METHODS: Thirty-six Sprague-Dawley rats received an open crush trauma of the left soleus muscle. One week after trauma, 2.5 x 10(6) autologous MSCs, harvested from tibial biopsies, were transplanted locally (female, n = 9; male, n = 9). Control animals received saline solution (female, n = 9; male, n = 9). Histologic analysis and biomechanical evaluation by in vivo muscle force measurement were performed 3 weeks after transplantation. RESULTS: MSC therapy improved the force of the injured soleus in male rats significantly (twitch: treated, 0.76 [0.51-1.15]; twitch: untreated, 0.45 [0.32-0.73] [P = 0.01]; tetany: treated, 0.63 [0.4-1.21], tetany: untreated, 0.34 [0.16-0.48] [P = 0.04]). Force measurements in females also revealed significant improvements (twitch: treated, 0.71 [0.38-0.96]; twitch: untreated, 0.36 [0.18-0.63] [P = 0.005]; tetany: treated, 0.53 [0.21-0.68]; tetany: untreated, 0.27 [0.11-0.47] [P = 0.01]). The intersexual comparison of fast twitch and tetanic contraction forces revealed no significance (twitch, P = 0.55; tetany, P = 0.19). The histologic analysis showed no differences in the amount of fibrotic tissue (male, P = 0.9; female, P = 0.14) and the size of muscle area (male, P = 0.2; female, P = 0.56) following treatment. Male animals showed higher values for muscle area (P = 0.011) and less fibrosis (P = 0.028), independent of treatment. CONCLUSION: The outcome of skeletal muscle regeneration after injury can be improved in animals of both sexes with MSC transplantation.
Abstract: Growth and differentiation factor 5 (GDF5), a member of the bone morphogenetic protein (BMP) family, is essential for cartilage, bone, and joint formation. Antagonists such as noggin counteract BMP signaling by covering the ligand's BMP type I (BMPRI) and type II (BMPRII, ActRII, ActRIIB) interaction sites. The mutation GDF5-S94N is located within the BMPRII interaction site, the so-called knuckle epitope, and was identified in patients suffering from multiple synostoses syndrome (SYNS). SYNS is characterized by progressive symphalangism, carpal/tarsal fusions, deafness and mild facial dysmorphism. Here we present a novel molecular mechanism of a GDF5 mutation affecting chondrogenesis and osteogenesis. GDF5-S94N exhibits impaired binding to BMPRII causing alleviated Smad and non-Smad signaling and reduced chondrogenic differentiation of ATDC5 cells. Surprisingly, chondrogenesis in mouse micromass cultures was strongly enhanced by GDF5-S94N. By using quantitative techniques (SPR, reporter gene assay, ALP assay, qPCR), we uncovered that this gain of function is caused by strongly reduced affinity of GDF5-S94N to the BMP/GDF antagonist noggin and the consequential lack of noggin inhibition. Thus, since noggin is upregulated during chondrogenic differentiation, GDF5-S94N exceeds the GDF5 action, which results in the phenotypic outcome of SYNS. The detailed molecular characterization of GDF5-S94N as a noggin-resistant growth factor illustrates the potential of GDF5 mutants in applications with defined therapeutical needs.
Abstract: Bone extracellular matrix consists of a network of proteins in which growth factors, like bone morphogenetic protein 2 (BMP-2), are embedded and released upon matrix turnover and degradation. Recombinant human (rh)BMP-2 shows promise in enhancing bone fracture repair, although issues regarding finding a suitable delivery system still limit its extensive clinical use. The aim of this study is to determine which cell activities are triggered by the presentation of immobilized rhBMP-2. For this purpose gold surfaces were first decorated with a self-assembled monolayer consisting of a hetero-bifunctional linker. rhBMP-2 was covalently bound to the surfaces via this linker and used to investigate the cellular responses of C2C12 myoblasts. We show that covalently immobilized rhBMP-2 (iBMP-2) initiates short-term signaling events. Using a BMP-responsive reporter gene assay and western blotting to monitor phosphorylation of Smad1/5/8 we prove that iBMP-2 activates BMP-dependent signal transduction. Furthermore, we demonstrate that iBMP-2 suppresses myotube formation and promotes the osteoblast phenotype in C2C12 cells. The bioactivity of surface-bound rhBMP-2 presented in this study is not due to its release into the medium. As such, our simple approach paves the way for the controlled local presentation of immobilized growth factors, limiting degradation while still maintaining biological activity.
Abstract: Chronic inflammatory diseases are characterized by hypoxia and subsequent cellular adaptation via hypoxia-inducible factor (HIF). Modulation of these adaptation mechanisms provides the basis for ideas on how to improve the effects of known drugs and for the development of novel therapeutic approaches.
Abstract: A novel method for non-destructive intramuscular fat (IMF) estimation via spectral ultrasound backscatter analysis of signals obtained from pig carcasses early post mortem is described. A commercial hand-held ultrasound device (center frequency: 2.7 MHz) was modified to focus the sound beam to the longissimus muscle at the 2nd/3rd last rib. Time-resolved ultrasound backscatter signals of loin muscle were recorded 45 min p.m. on 82 pig carcass sides. Backfat width (d(BF)=18.9+/-3.8 mm) and muscle attenuation (alpha(muscle)=.77+/-.15 dB MHz(-1) cm(-1)) were assessed from the measured pulse echo data. Other propagation properties of skin, backfat and muscle tissue obtained in a previous investigation were incorporated into the signal pre-processing to minimize parameter estimation artifacts. Spectral and cepstral parameters were derived from time-gated backscattered signals measured in the central muscle region. The range of intramuscular fat (IMF) determined by ether extraction was representative for German pig populations (.7%</=IMF(chem)</=3.6%, coefficient of variation CV(IMF(chem))=44.8%). Variations of IMF were associated with variations of backfat width (CV(d(BF))=20.2%), muscle attenuation (CV(alpha(muscle))=19.3%), and slope of the backscattered amplitude spectrum (CV(m)=28.8%). A full cross validated multiple linear regression model using these parameters resulted in good predictability of IMF(chem) (R(2)=.76, RMSEP=.34%). Among all tested carcasses, 73% could be correctly classified into one of three IMF classes (LOW: <1%, MID: 1-2%, HIGH: >2%). Using a single threshold (2% IMF), about 92% of all carcasses were correctly classified. With respect to the inherent variability of IMF within a single muscle and the different tissue volumes used for the chemical and ultrasound based IMF estimations the remaining prediction errors are acceptable. Compared to previous ultrasound based studies, the number of acoustic parameters used for the IMF prediction could be reduced. Moreover, the used parameters are based on time-of-flight and spectral slope estimations, which are i) more robust with respect to measurement artifacts and ii) have a causal link to structural variations associated with IMF variations in pork loin.
Abstract: The first successful reprogramming of differentiated cells to a pluripotent state was done by retroviral introduction of four transcription factors (Oct4, Sox2, Klf4, cMyc) by the group of Yamanaka in 2006. Since then, scientists all over the world have attempted various methods to avoid insertional mutagenesis, a major limitation of the retrovirus-based method, however no technique was found to completely avoid DNA integration. Recently, a non-viral mRNA-based approach, inherent to avoid genomic integration, was implemented to generate stem cell-like cells, yet, seventeen daily transfections were required, inducing substantial stress on the cells. In this work, we demonstrate successful activation of pluripotency-associated genes in mouse embryonic fibroblasts by means of cationic lipid-mediated introduction of mRNAs encoding the four factors. Moreover, our transfection protocol required maximally three transfections. Up-regulation of the transfected factors as well as Nanog and SSEA-1, typical mouse pluripotency markers, was detected already after the first transfection. Nuclear localization of the introduced factors was confirmed. Positive alkaline phosphatase staining of cell clusters further confirmed the onset of the reprogramming process. In conclusion, the transfection method presented here holds great promise for safe generation of induced pluripotent stem cells of mouse origin.
Abstract: Ligands of the Transforming Growth Factor beta superfamily like Transforming Growth Factor beta and Bone Morphogenetic Proteins govern developmental processes and regulate adult homeostasis by controlling cellular proliferation, survival, differentiation and migration. Aberrant signalling activity is associated with human disorders such as cancer, cardiovascular, musculoskeletal, or fibrotic disease. Upon binding to specific sets of cognate cell surface receptors, family members induce highly similar pathways which include canonical SMAD dependent signalling as well as pathways without direct involvement of SMAD proteins, which activate signalling molecules like mitogen-activated protein kinases or small GTPases. The diverse ligand functionalities are achieved through regulation and modulation of the pathways at all levels, resulting in a highly quantitative and context sensitive signal integration reflecting the cellular state and background. Strategies to target Transforming Growth Factor beta or Bone Morphogenetic Protein pathways have been developed on the basis of our current understanding and have proven a highly beneficial potential.
Abstract: Delivery of reprogramming factor-encoding mRNAs by means of lipofection in somatic cells is a desirable method for deriving integration-free iPSCs. However, the lack of reproducibility implies there are major hurdles to overcome before this protocol becomes universally accepted. This study demonstrates the functionality of our in-house synthesized mRNAs expressing the reprogramming factors (OCT4, SOX2, KLF4, c-MYC) within the nucleus of human fibroblasts. However, upon repeated transfections, the mRNAs induced severe loss of cell viability as demonstrated by MTT cytotoxicity assays. Microarray-derived transcriptome data revealed that the poor cell survival was mainly due to the innate immune response triggered by the exogenous mRNAs. We validated the influence of mRNA transfection on key immune response-associated transcript levels, including IFNB1, RIG-I, PKR, IL12A, IRF7 and CCL5, by quantitative real-time PCR and directly compared these with the levels induced by other methods previously published to mediate reprogramming in somatic cells. Finally, we evaluated chemical compounds (B18R, chloroquine, TSA, Pepinh-TRIF, Pepinh-MYD), known for their ability to suppress cellular innate immune responses. However, none of these had the desired effect. The data presented here should provide the basis for further investigations into other immunosuppressing strategies that might facilitate efficient mRNA-mediated cellular reprogramming in human cells.
Abstract: An immense number of cellular processes are initiated by cell surface serine/threonine kinase receptors belonging to the TGF-beta/BMP family. Subsequent downstream signalling cascades, as well as their crosstalk results in enormous specificity in terms of phenotypic outcome, e.g. proliferation, differentiation, migration or apoptosis. Such signalling diversity is achieved by the ability of receptors to interact with distinct proteins in a spatio-temporal manner. Following the cloning of the TGF-beta/BMP receptors a variety of different technologies were applied to identify such interacting proteins. Here we present a comprehensive survey of known interactome analyses, including our own data, on these receptors and discuss advantages and disadvantages of the applied technologies.
Abstract: A major clinical problem of high relevance in the cardiovascular field is late stent thrombosis after implantation of drug eluting stents (DES). Clinical widely used DES currently utilize durable polymer coatings, which can induce persistent arterial wall inflammation and delayed vascular healing resulting in an impaired endothelialization. In this study we explored the interaction of smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC) with electrospun scaffolds prepared from resorbable polyetheresterurethane (PDC) and poly(p-dioxanone) (PPDO), as well as polyetherimide (PEI), which can be surface modified, in comparison to poly(vinylidene fluoride-co-hexafluoropropene) (PVDF) as reference material, which is established as coating material of DES in clinical applications. Our results show that adhesion could be improved for HUVEC on PDC, PPDO and PEI compared to PVDF, whereas almost no SMC attached to the scaffolds indicating a cell-specific response of HUVEC towards the different fibrous structures. Proliferation and apoptosis results revealed that PPDO and PEI have no significant negative influence on vitality and cell cycle behaviour compared to PVDF. Hence, they represent promising candidates for temporary blood vessel support that induce HUVEC attachment and prevent SMC proliferation.
Abstract: IL-17-producing CD4+ T helper cells (TH17) have been extensively investigated in mouse models of autoimmunity. However, the requirements for differentiation and the properties of pathogen-induced human TH17 cells remain poorly defined. Using an approach that combines the in vitro priming of naive T cells with the ex vivo analysis of memory T cells, we describe here two types of human TH17 cells with distinct effector function and differentiation requirements. Candida albicans-specific TH17 cells produced IL-17 and IFN-gamma, but no IL-10, whereas Staphylococcus aureus-specific TH17 cells produced IL-17 and could produce IL-10 upon restimulation. IL-6, IL-23 and IL-1beta contributed to TH17 differentiation induced by both pathogens, but IL-1beta was essential in C. albicans-induced TH17 differentiation to counteract the inhibitory activity of IL-12 and to prime IL-17/IFN-gamma double-producing cells. In addition, IL-1beta inhibited IL-10 production in differentiating and in memory TH17 cells, whereas blockade of IL-1beta in vivo led to increased IL-10 production by memory TH17 cells. We also show that, after restimulation, TH17 cells transiently downregulated IL-17 production through a mechanism that involved IL-2-induced activation of STAT5 and decreased expression of ROR-gammat. Taken together these findings demonstrate that by eliciting different cytokines C. albicans and S. aureus prime TH17 cells that produce either IFN-gamma or IL-10, and identify IL-1beta and IL-2 as pro- and anti-inflammatory regulators of TH17 cells both at priming and in the effector phase.
Abstract: A major clinical problem of high relevance in the cardiovascular field is late stent thrombosis after implantation of drug eluting stents (DES). Clinical widely used DES currently utilize durable polymer coatings, which can induce persistent arterial wall inflammation and delayed vascular healing resulting in an impaired endothelialization. In this study we explored the interaction of smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC) with electrospun scaffolds prepared from resorbable polyetheresterurethane (PDC) and poly(p-dioxanone) (PPDO), as well as polyetherimide (PEI), which can be surface modified, in comparison to poly(vinylidene fluoride-co-hexafluoropropene) (PVDF) as reference material, which is established as coating material of DES in clinical applications. Our results show that adhesion could be improved for HUVEC on PDC, PPDO and PEI compared to PVDF, whereas almost no SMC attached to the scaffolds indicating a cell-specific response of HUVEC towards the different fibrous structures. Proliferation and apoptosis results revealed that PPDO and PEI have no significant negative influence on vitality and cell cycle behaviour compared to PVDF. Hence, they represent promising candidates for temporary blood vessel support that induce HUVEC attachment and prevent SMC proliferation.
Abstract: A year-long intervention trial was conducted to characterise the responses of multiple biomarkers of Se status in healthy American adults to supplemental selenomethionine (SeMet) and to identify factors affecting those responses. A total of 261 men and women were randomised to four doses of Se (0, 50, 100 or 200 mug/d as l-SeMet) for 12 months. Responses of several biomarkers of Se status (plasma Se, serum selenoprotein P (SEPP1), plasma glutathione peroxidase activity (GPX3), buccal cell Se, urinary Se) were determined relative to genotype of four selenoproteins (GPX1, GPX3, SEPP1, selenoprotein 15), dietary Se intake and parameters of single-carbon metabolism. Results showed that supplemental SeMet did not affect GPX3 activity or SEPP1 concentration, but produced significant, dose-dependent increases in the Se contents of plasma, urine and buccal cells, each of which plateaued by 9-12 months and was linearly related to effective Se dose (mug/d per kg0.75). The increase in urinary Se excretion was greater for women than men, and for individuals of the GPX1 679 T/T genotype than for those of the GPX1 679 C/C genotype. It is concluded that the most responsive Se-biomarkers in this non-deficient cohort were those related to body Se pools: plasma, buccal cell and urinary Se concentrations. Changes in plasma Se resulted from increases in its non-specific component and were affected by both sex and GPX1 genotype. In a cohort of relatively high Se status, the Se intake (as SeMet) required to support plasma Se concentration at a target level (Sepl-target) is: Se_{in} = [(Se_{pl - target} - Se_{pl})/(18.2\hairsp ng\,d\,kg^{0.75}/ml\,per\,\mu g)] .
Abstract: Effector memory T cells are effective in controlling acute infections, but central memory T cells play a key role in long-lasting protection against viruses and tumors. In vivo/in vitro challenge by Ag commonly supports the generation of effector memory T cells with limited longevity. To our knowledge, this study demonstrates for the first time in the human system and under rechallenge conditions that targeting IL-2R by partial mammalian target of rapamycin inhibition or blocking IL-2Ralpha enriches human CD4(+)/CD8(+) central memory T cells within the virus-specific T cell product associated with enhanced functionality (i.e., multicytokine secretors, including IL-2; enhanced CD137 and CD107a expression on CD8(+) and CD4(+) T cells, respectively; and killing infected target cells). Remarkably, the effects on CD8(+) T cells are mainly mediated via the enhancement of CD4(+) T cell function. The data reveal new insights into the role of CD4(+) T cell support for the quality of CD8(+) T cell memory, even under rechallenge conditions. Moreover, our method offers a new approach to improve the long-lasting efficacy of adoptive T cell therapy in patients.
Abstract: The shape-memory properties of electrospun polyetherurethanes (PEU) non-wovens with a single fiber diameter of around 1 μm were explored. In uniaxial cyclic, thermomechanical tensile tests a dual-shape shape-memory creation procedure (SMCP) was applied and the shape recovery was examined under stress-free and constant strain conditions. The thermal properties of the electrospun PEU non-wovens were found to be similar to those obtained for bulk PEU samples, whereas the mechanical properties revealed differences with respect to the elongation at break (εb) at increased temperatures. Excellent dual-shape properties were achieved for the PEU non-wovens with a high shape fixity rate (Rf) and shape recovery rate (Rr). A significant higher recovery stress (σmax) was obtained under constant strain recovery conditions for the electrospun non-wovens compared to the bulk PEU samples, which might be attributed to the higher degree of orientation of the polymer chains in the microfibers. Therefore the influence of different (single) fiber diameters as well as the variation of the programming elongation εm and temperature Tprog on σmax is an interesting issue for future investigations.
Abstract: We identified an amino acid change (p.G92E) in the Bone Morphogenetic Protein antagonist NOGGIN in a 22-month-old boy who presented with a unilateral brachydactyly type B phenotype. Brachydactyly type B is a skeletal malformation that has been associated with increased Bone Morphogenetic Protein pathway activation in other patients. Previously, the amino acid change p.G92E in NOGGIN was described as causing fibrodysplasia ossificans progressiva, a rare genetic disorder characterized by limb malformations and progressive heterotopic bone formation in soft tissues that, like Brachydactyly type B, is caused by increased activation of Bone Morphogenetic Protein signaling. To determine whether G92E-NOGGIN shows impaired antagonism that could lead to increased Bone Morphogenetic Protein signaling, we performed functional assays to evaluate inhibition of BMP signaling. Interestingly, wt-NOGGIN shows different inhibition efficacies towards various Bone Morphogenetic Proteins that are known to be essential in limb development. However, comparing the biological activity of G92E-NOGGIN with wt-NOGGIN, we observed that G92E-NOGGIN inhibits activation of bone morphogenetic protein signaling with equal efficiency as wt-NOGGIN, supporting that G92E-NOGGIN does not cause pathological effects. Genetic testing of the child's parents revealed the same amino acid change in the healthy father, further supporting that p.G92E is a neutral amino acid substitution in NOGGIN. We conclude that p.G92E represents a rare polymorphism of the NOGGIN gene - causing neither brachydactyly nor fibrodysplasia ossificans progressiva. This study highlights that a given genetic variation should not be considered pathogenic unless supported by functional analyses.
Abstract: The musculoskeletal system is a tight network of many tissues. Coordinated interplay at a biochemical level between tissues is essential for development and repair. Traumatic injury usually affects several tissues and represents a large challenge in clinical settings. The current demand for potent growth factors in such applications thus accompanies the keen interest in molecular mechanisms and orchestration of tissue formation. Of special interest are multitasking growth factors that act as signals in a variety of cell types, both in a paracrine and in an autocrine manner, thereby inducing cell differentiation and coordinating not only tissue assembly at specific sites but also maturation and homeostasis. We concentrate here on bone morphogenetic proteins (BMPs), which are important crosstalk mediators known for their irreplaceable roles in vertebrate development. The molecular crosstalk during embryonic musculoskeletal tissue formation is recapitulated in adult repair. BMPs act at different levels from the initiation to maturation of newly formed tissue. Interestingly, this is influenced by the spatiotemporal expression of different BMPs, their receptors and co-factors at the site of repair. Thus, the regenerative potential of BMPs needs to be evaluated in the context of highly connected tissues such as muscle and bone and might indeed be different in more poorly connected tissues such as cartilage. This highlights the need for an understanding of BMP signaling across tissues in order to eventually improve BMP regenerative potential in clinical applications. In this review, the distinct members of the BMP family and their individual contribution to musculoskeletal tissue repair are summarized by focusing on their paracrine and autocrine functions.
Abstract: The pore structures of microparticulate drug carriers are important diffusion pathways, which are not a static property but rather may be changing in the case of degradable matrix polymers such as poly[(rac-lactide)-co-glycolide] (PLGA). In this study, the mutual impacts of dynamic changes in microparticle porosity and polymer degradation were analyzed for PLGA with different molecular weights and end groups as well as PLGA-based triblock copolymers. In selected cases, particularly for PLGA with hydrophilic end groups and low initial number average molecular weight of 5 kDa, pore opening/pore closing phenomena were detected during incubation in phosphate buffer at 37 °C. Initially, pore closing was induced by water-induced plasticization and the reduction of interfacial tension. The pattern of molecular weight decrease and mass loss suggested that pore closing did not result in undesired autocatalytic acceleration of degradation or delayed mass loss due to trapped acidic degradation products.
Abstract: The type I and type II bone morphogenetic protein receptors (BMPRI and BMPRII) are present at the plasma membrane as monomers and homomeric and heteromeric complexes, which are modulated by ligand binding. The complexes of their extracellular domains with ligand were shown to form heterotetramers. However, the dynamics of the oligomeric interactions among the full-length receptors in live cell membranes were not explored, and the roles of BMP receptor homodimerization were unknown. Here, we investigated these issues by combining patching/immobilization of an epitope-tagged BMP receptor at the cell surface with measurements of the lateral diffusion of a co-expressed, differently tagged BMP receptor by fluorescence recovery after photobleaching (FRAP). These studies led to several novel conclusions. (a) All homomeric complexes (without or with BMP-2) were stable on the patch/FRAP time scale (minutes), whereas the heterocomplexes were transient, a difference that may affect signaling. (b) Patch/FRAP between HA- and myc-tagged BMPRII combined with competition by untagged BMPRIb showed that the heterocomplexes form at the expense of homodimers. (c) Stabilization of BMPRII·BMPRIb heterocomplexes (but not homomeric complexes) by IgG binding to same-tag receptors elevated phospho-Smad formation both without and with BMP-2. These findings suggest two mechanisms that may suppress the tendency of preformed BMP receptor hetero-oligomers to signal without ligand: (a) competition between homo- and heterocomplex formation, which reduces the steady-state level of the latter, and (b) the transient nature of the heterocomplexes, which limits the time during which BMPRI can be phosphorylated by BMPRII in the heterocomplex.
Abstract: Collagen is a versatile structural molecule in nature and is used as a building block in many highly organized tissues, such as bone, skin, and cornea. The functionality and performance of these tissues are controlled by their hierarchical organization ranging from the molecular up to macroscopic length scales. In the present study, polarized Raman microspectroscopic and imaging analyses were used to elucidate collagen fibril orientation at various levels of structure in native rat tail tendon under mechanical load. In situ humidity-controlled uniaxial tensile tests have been performed concurrently with Raman confocal microscopy to evaluate strain-induced chemical and structural changes of collagen in tendon. The methodology is based on the sensitivity of specific Raman scattering bands (associated with distinct molecular vibrations, such as the amide I) to the orientation and the polarization direction of the incident laser light. Our results, based on the changing intensity of Raman lines as a function of orientation and polarization, support a model where the crimp and gap regions of collagen hierarchical structure are straightened at the tissue and molecular level, respectively. However, the lack of measurable changes in Raman peak positions throughout the whole range of strains investigated indicates that no significant changes of the collagen backbone occurs with tensing and suggests that deformation is rather redistributed through other levels of the hierarchical structure.
Abstract: Degradable polymers are essential to enable biomaterial-based regenerative therapies, particularly, in articular cartilage defect healing, which remains a major clinical challenge. The aim of this study was to investigate the effect of two degradable polymers (as a film or scaffold) on primary articular chondrocytes vitality, adherence, differentiated phenotype and morphology. Films and electro-spun scaffolds were prepared from degradable poly(ether)ester urethane (PDC), which was synthesized via co-condensation of poly(p-dioxanone)diol and poly(ε-caprolactone)diol with an aliphatic diisocyanate and poly(p-dioxanone) (PPDO). The thermal and mechanical properties and the surface roughness of the films and scaffolds were examined by differential scanning calorimetry, dynamic mechanical thermal analysis, tensile tests and optical profilometry. Primary porcine articular chondrocytes were seeded on the polymers and analysed for viability, ultrastructure (scanning electron microscopy) and immunolabelled for type II collagen. All films and scaffolds exhibited a low endotoxin load < 0.06 EU/ml and only moderate cytotoxic effects when tested with L929 cells. The results of the seeding experiments revealed that survival and adhesion of chondrocytes depended strongly on seeding density. Vital chondrocytes could be detected on both PPDO and PDC films and scaffolds. They produced the cartilage-specific protein type II collagen indicating differentiated functions. However, they exhibited a mixed morphology on the films and a more flattened cell shape on the scaffolds. The cell/biomaterial interaction in the PPDO scaffolds or films was more intense compared to that in PDC topologies. xD; xD;The polymer topology influences chondrocytes phenotype, whereby the scaffolds permitted a more continuous cell growth compared with the films.
Abstract: Somatic cells can be reprogrammed to induced pluripotent stem cells by over-expression of OCT4, SOX2, KLF4 and c-MYC (OSKM). With the aim of unveiling the early mechanisms underlying the induction of pluripotency, we have analyzed transcriptional profiles at 24, 48 and 72 hours post-transduction of OSKM into human foreskin fibroblasts. Experiments confirmed that upon viral transduction, the immediate response is innate immunity, which induces free radical generation, oxidative DNA damage, p53 activation, senescence, and apoptosis, ultimately leading to a reduction in the reprogramming efficiency. Conversely, nucleofection of OSKM plasmids does not elicit the same cellular stress, suggesting viral response as an early reprogramming roadblock. Additional initiation events include the activation of surface markers associated with pluripotency and the suppression of epithelial-to-mesenchymal transition. Furthermore, reconstruction of an OSKM interaction network highlights intermediate path nodes as candidates for improvement intervention. Overall, the results suggest three strategies to improve reprogramming efficiency employing: 1) anti-inflammatory modulation of innate immune response, 2) pre-selection of cells expressing pluripotency-associated surface antigens, 3) activation of specific interaction paths that amplify the pluripotency signal.
Abstract: Metabologens initiate, promote and maintain morphogenesis and adult tissue homeostasis. Bone morphogenetic proteins (BMPs) which belong to the transforming growth factor-[beta] (TGF-[beta]) superfamily, represent a major class of metabologens that regulate ectoderm, mesoderm and endoderm derived tissue formation. In order to temporally and spatially control BMP initiated signaling cascades, a tight regulatory network is needed to maintain concinnity. There are a number of ways how BMP signaling can be inhibited or more likely be modified, among which the direct extracellular inhibition through cysteine-knot containing proteins from the DAN-, the twisted gastrulation-, chordin- and noggin-family is a classic. This review focuses on noggin and its impact on the vast array of BMP driven actions and thereby invites the ever-growing BMP research field to (re-) investigate noggin's function in detail.
Abstract: It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.
Abstract: Polyetherimides have been evaluated as non-toxic and steam-sterilizable and are therefore potentially suited for biomedical applications. To enable a broader range of potential applications, polyetherimides with lower Tg, higher elasticity at room temperature and better processability are required. Our concept was to explore, whether the incorporation of 3-methoxy-1,2-propanediol moieties in the main chain lead to a reduction of Tg and increase the elastic properties of the polymer compared to commercially available polyetherimides from 4,4′-(4,4′-isopropylidenediphenoxy)bis(phthalic anhydride) and 1,3-diaminobenzene. Two different monomers were synthesized and co-condensated with each other or using 4,4,4′-(4,4′-isopropylidenediphenoxy)bis(phthalic anhydride), respectively. The results proofed the successful synthesis and polymerization leading to polymers with molecular weights up to Mn = 6,400 g/mol. The polymers showed lowered Tg, resistance to heat up to 400 °C, tendencies to reduced contact angles and partially reduced E-moduli in comparison to the commercial polyetherimide ULTEM 1000.
Abstract: Across many tissues and organs, the ability to create an organoid, the smallest functional unit of an organ, in vitro is the key both to tissue engineering and preclinical testing regimes. The hair follicle is an organoid that has been much studied based on its ability to grow quickly and to regenerate after trauma. But hair follicle formation in vitro has been elusive. Replacing hair lost due to pattern baldness or more severe alopecia, including that induced by chemotherapy, remains a significant unmet medical need. By carefully analyzing and recapitulating the growth conditions of hair follicle formation, we recreated human hair follicles in tissue culture that were capable of producing hair. Our microfollicles contained all relevant cell types and their structure and orientation resembled in some ways excised hair follicle specimens from human skin. This finding offers a new window onto hair follicle development. Having a robust culture system for hair follicles is an important step towards improved hair regeneration as well as to an understanding of how marketed drugs or drug candidates, including cancer chemotherapy, will affect this important organ.
Abstract: Introduction  The objectives of this study were to (1) establish a reproducible atrophic non-union model in rats by creation of a segmental femoral bone defect that allows, (2) in-depth characterization of impaired healing, and (3) contrast its healing patterns to the normal course. Hypothesis was that a 5-mm bone defect in male rats would deviate from uneventful healing patterns and result in an atrophic non-union. Materials and methods  A femoral osteotomy was performed in two groups of 12-week-old male rats (1 vs. 5 mm gap) stabilized with an external fixator. Bone healing in these models was evaluated by radiology, biomechanics, and histology at 6 or 8 weeks. The evaluation of the 5-mm group revealed in some cases a delayed rather than a non-union, and therefore, a group of female counterparts was included. Results  The creation of a 5-mm defect in female rats resulted in a reproducible atrophic non-union characterized by sealing of the medullary canal, lack of cartilage formation, and negligible mechanical properties of the callus. In both gap size models, the male subjects showed advanced healing compared to females. Discussion and conclusion  This study showed that even under uneventful healing conditions in terms of age and bone defect size, there is a sex-specific advanced healing in male compared to female subjects. Contrary to our initial hypothesis, only the creation of a 5-mm segmental femoral defect in female rats led to a reproducible atrophic non-union. It has been shown that an atrophic non-union exhibits different healing patterns compared to uneventful healing. A total lack of endochondral bone formation, soft tissue prolapse into the defect, and bony closure of the medullary cavity have been shown to occur in the non-union model.
Abstract: e assumption that mesenchymal stromal cell (MSC)-based-therapies are capable of augmenting physiological regeneration processes has fostered intensive basic and clinical research activities. However, to achieve sustained therapeutic success in vivo, not only the biological, but also the mechanical microenvironment of MSCs during these regeneration processes needs to be taken into account. This is especially important for e.g., bone fracture repair, since MSCs present at the fracture site undergo significant biomechanical stimulation. This study has therefore investigated cellular characteristics and the functional behaviour of MSCs in response to mechanical loading. Our results demonstrated a reduced expression of MSC surface markers CD73 (ecto-5'-nucleotidase) and CD29 (integrin beta1) after loading. On the functional level, loading led to a reduced migration of MSCs. Both effects persisted for a week after the removal of the loading stimulus. Specific inhibition of CD73/CD29 demonstrated their substrate dependent involvement in MSC migration after loading. These results were supported by scanning electron microscopy images and phalloidin staining of actin filaments displaying less cell spreading, lamellipodia formation and actin accumulations. Moreover, focal adhesion kinase and Src-family kinases were identified as candidate downstream targets of CD73/CD29 that might contribute to the mechanically induced decrease in MSC migration. These results suggest that MSC migration is controlled by CD73/CD29, which in turn are regulated by mechanical stimulation of cells. We therefore speculate that MSCs migrate into the fracture site, become mechanically entrapped, and thereby accumulate to fulfil their regenerative functions.
Abstract: Somatic cells reprogrammed into induced pluripotent stem cells (iPSCs) acquire features of human embryonic stem cells (hESCs) and thus represent a promising source for cellular therapy of debilitating diseases, such as age-related disorders. However, reprogrammed cell lines have been found to harbor various genomic alterations. In addition, we recently discovered that the mitochondrial DNA of human fibroblasts also undergoes random mutational events upon reprogramming. Aged somatic cells might possess high susceptibility to nuclear and mitochondrial genome instability. Hence, concerns over the oncogenic potential of reprogrammed cells due to the lack of genomic integrity may hinder the applicability of iPSC-based therapies for age-associated conditions. Here, we investigated whether aged reprogrammed cells harboring chromosomal abnormalities show resistance to apoptotic cell death or mitochondrial-associated oxidative stress, both hallmarks of cancer transformation. Four iPSC lines were generated from dermal fibroblasts derived from an 84-year-old woman, representing the oldest human donor so far reprogrammed to pluripotency. Despite the presence of karyotype aberrations, all aged-iPSCs were able to differentiate into neurons, re-establish telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a hESC-like state. Importantly, aged-iPSCs exhibited high sensitivity to drug-induced apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and hESCs. Thus, the occurrence of chromosomal abnormalities within aged reprogrammed cells might not be sufficient to over-ride the cellular surveillance machinery and induce malignant transformation through the alteration of mitochondrial-associated cell death. Taken together, we unveiled that cellular reprogramming is capable of reversing aging-related features in somatic cells from a very old subject, despite the presence of genomic alterations. Nevertheless, we believe it will be essential to develop reprogramming protocols capable of safeguarding the integrity of the genome of aged somatic cells, before employing iPSC-based therapy for age-associated disorders.
Abstract: Human induced pluripotent stem cells (iPSCs) have been recently found to harbor genomic alterations. However, the integrity of mitochondrial DNA (mtDNA) within reprogrammed cells has yet to be investigated. mtDNA mutations occur at a high rate and contribute to the pathology of a number of human disorders. Furthermore, the lack of mtDNA integrity may alter cellular bioenergetics and limit efficient differentiation. We demonstrated previously that the derivation of iPSCs is associated with mitochondrial remodeling and a metabolic switch towards glycolysis. Here, we have discovered that alterations of mtDNA can occur upon the induction of pluripotency. Massively parallel pyrosequencing of mtDNA revealed that human iPSCs derived from young healthy donors harbored single base mtDNA mutations (substitutions, insertions, and deletions), both homoplasmic (in all mtDNA molecules) and heteroplasmic (in a fraction of mtDNAs), not present in the parental cells. mtDNA modifications were mostly common variants and not disease related. Moreover, iPSC lines bearing different mtDNA mutational loads maintained a consistent human embryonic stem cell-like reprogramming of energy metabolism. This involved the upregulation of glycolytic enzymes, increased glucose-6-phosphate levels, and the over-expression of pyruvate dehydrogenase kinase 1 protein, which reroutes the bioenergetic flux toward glycolysis. Hence, mtDNA mutations within iPSCs may not necessarily impair the correct establishment of pluripotency and the associated metabolic reprogramming. Nonetheless, the occurrence of pathogenic mtDNA modifications might be an important aspect to monitor when characterizing iPSC lines. Finally, we speculate that this random rearrangement of mtDNA molecules might prove beneficial for the derivation of mutation-free iPSCs from patients with mtDNA disorders.
Abstract: Inflammatory resolution is an active, highly regulated process already encoded at the onset of inflammation and required to prevent the transition into chronic inflammation associated with spreading of tissue injury and exacerbated scarring. We introduce objective, quantitative measurements [resolution indices (Ri) and resolution plateau (RP)] to characterize inflammatory resolution and to determine the persistence (“dwell timeâ€) of differential leukocyte subpopulations at the lesion site after acute experimental spinal cord injury (SCI). The cell type-specific resolution interval Ri (time between maximum cell numbers and the point when they are reduced to 50%) ranges from 1.2 days for neutrophils, 1.5 days for T lymphocytes, to 55 days for microglia/macrophages. As the resolution interval neglects exiting cell trafficking in the later period of resolution (49%–0% of lesional cells), we introduced the RP, a marker for the persisting, chronified leukocyte subsets, which are likely to participate in late degeneration and non-resolving inflammation. Here, we identify the acute inflammatory response in central nervous system (CNS) lesions as partly non self-limiting. Both extended resolution intervals (reduced leukocyte clearance) and elevated plateaus (permanent lesional cell numbers) provide quantitative measures to characterize residual, sustained inflammation and define cognate timeframes of impaired resolution after acute SCI.
Abstract: Objective The objective was to study the associations between serum selenium concentration and thyroid volume, as well as the association between serum selenium concentration and risk for an enlarged thyroid gland in an area with mild iodine deficiency before and after iodine fortification was introduced. Another objective was to examine the association between serum selenium concentration and prevalence of thyroid nodules.Design Cross-sectional study.Methods We studied participants of two similar cross-sectional studies carried out before (1997–1998, n=405) and after (2004–2005, n=400) introduction of iodine fortification. Serum selenium concentration and urinary iodine were measured, and the thyroid gland was examined by ultrasonography in the same subjects. Associations between serum selenium concentration and thyroid parameters were examined in multiple linear regression models or logistic regression models.Results Serum selenium concentration was found to be significantly, negatively associated with thyroid volume (P=0.006), and a low selenium status significantly increased the risk for thyroid enlargement (P=0.007). Furthermore, low serum selenium status had a tendency to increase the risk for development of multiple nodules (P=0.087).Conclusions Low serum selenium concentration was associated with a larger thyroid volume and a higher prevalence of thyroid enlargement.
Abstract: The volume of the gluteus medius muscle (GMV) has been shown to be closely related to hip joint function. A defined relation between joint geometry and GMV would allow a calculation of the patient-specific GMV providing reference values to individually determine the goals for rehabilitation programs after total hip arthroplasty (THA). The aim of this study was to investigate correlations between hip geometry and GMV. One hundred and two (50 female, age: 58.53 (18-86)) pelvic computed tomography (CT) scans were analyzed to determine femoral offset (FO), body weight lever arm (BWLA) and the GMV. Relationships between demographic data, FO and GMV were analyzed using correlation and regression analysis. The mean GMV was found to be 289 ± 72 cm3, the mean FO measured was 4.14 ± 0.55 cm; and the mean value for BWLA measured was 8.88 ± 0.4 cm. A formula to calculate the GMV with a good coefficient of determination (R2 = 0.681) (p < 0.0001) was derived. In conclusion, the formula obtained predicts individual GMV with good model fit and could be used to individually determine rehabilitation goals. Moreover, the correlation found could account for a hand in hand development of FO and GMV during growth and a continuous functional relationship thereafter.
Abstract: The course of bone healing in animal models is conventionally monitored by morphologic approaches, which do not allow the determination of the material properties of the tissues involved. Mechanical characterization techniques are either dedicated to the macroscopic evaluation of the entire organ or to the microscopic evaluation of the tissue matrix. The latter provides insight to regionally specific alterations at the tissue level in the course of healing. In this study, quantitative scanning acoustic microscopy was used at 50 MHz to investigate microstructural and elastic alterations of mineralized callus and cortical tissue after transverse osteotomy in sheep tibiae. Analyses were performed after 2, 3, 6 and 9 weeks of consolidation with stabilization by either a rigid or a semi-rigid external fixator. Increased stiffness and decreased porosity were observed in the callus tissue over the course of the healing process, which was dependent on the fixator type. In the adjacent cortical tissue, stiffness decreased during the first 3 weeks. Cortical porosity increased over time but the time-dependence was different between the two fixator types. The changes of stiffness of cortical and callus tissues were measured with respect to the distance to the periosteal cortex-callus boundary. Stiffness of cortex and callus tissue smoothly decreased as a function of the distance from the inner cortical region. The data obtained in this study can help to understand the processes involved in tissue maturation during endogenous bone healing. (E-mail: kay.raum@charite.de)
Abstract: OBJECTIVE: Glucocorticoids (GCs) exert their antiinflammatory and immunosuppressive effects in humans primarily via the cytosolic GC receptor (cGR) but also via rapid, nongenomic mechanisms. Most likely, membrane-bound GRs (mGR) are involved in nongenomic GC signaling. The aim of this study was to investigate the origin and functional activity of mGR. METHODS: We analyzed the origin of mGR using mGR-expressing HEK 293T cells, by transient and stable RNA interference-mediated GR reduction. GR messenger RNA (mRNA) and cGR and mGR protein levels were analyzed by real-time quantitative polymerase chain reaction, immunoblotting, and high-sensitivity immunofluorescence staining. Furthermore, we analyzed the functional activity of mGR, using membrane-impermeable bovine serum albumin (BSA)-bound dexamethasone (DEX-BSA) in human monocytes. Membrane-bound GR-expressing monocytes were treated with DEX, DEX-BSA, or BSA. Cell lysates were analyzed using PepChip arrays in order to identify kinases triggered by DEX-BSA, with validation using Bio-Plex assays and immunoblotting. RESULTS: Our data showed that transient reduction of GR mRNA in HEK 293T cells decreased cGR protein levels but not mGR protein levels. However, stably transfected cells showed reduced cGR protein expression and significantly reduced mGR protein expression. Furthermore, 51 kinase substrates were identified for which phosphorylation was either reduced or increased. We observed p38 MAP kinase (MAPK) as one possible upstream kinase. Validation of these data by Bio-Plex phosphoprotein assay and immunoblotting showed increased phosphorylation of p38 MAPK after treatment with DEX-BSA. CONCLUSION: Our data demonstrate that the human GR gene encodes for both cGR and mGR. Membrane-bound GR retains functional activity, as indicated by induced phosphorylation of p38 MAPK due to DEX-BSA treatment. Membrane-bound GR-mediated cellular signaling needs to be investigated further in order to clarify its therapeutic potential.
Abstract: BACKGROUND: Polyomavirus BK virus (BKV) infection represents a serious complication leading to BKV-associated nephropathy (BKVAN) and subsequent kidney graft loss in up to 10% of transplant patients. Cellular immunity is known to play a crucial role in the control of BKV replication. However, the knowledge on the BKV-T-cell response is limited: only two (VP1 and large T antigen) of six known BKV proteins were evaluated for their antigenicity so far. METHODS: By using 10-color flow cytometry and newly created overlapping peptide pools of five BKV antigens (VP1, VP2, VP3, large T antigen, and small t antigen), we performed cross-sectional phenotypic and functional analysis of BKV-specific T cells in kidney transplant patients with a history of BKVAN. Patients with clinically unapparent BKV infection (history of transient/no BKV reactivation) were used as control group. RESULTS: Our data demonstrate for the first time the antigenic properties of all five evaluated proteins with VP3 as a new important target of cellular immunity. Further, we found a correlation between the severity of the previous BKV infection and the magnitude of memory CD4+ T-cell response. Thus, compared with the control group, patients with a history of BKVAN demonstrated significantly higher frequencies of interferon-gamma- and interleukin-2-producing effector memory CD4+ T cells. In the control group, more patients with detectable interferon-gamma+/interleukin-2+/tumor necrosis factor+ triple producers were found, suggesting possibly a protective function of these multifunctional T cells. CONCLUSIONS: In conclusion, our study results suggest an implementation of new targets for monitoring of BKV immunity. Further studies are required to evaluate the protective function of the found BKV-specific T-cell subsets.
Abstract: Mesenchymal stromal cells (MSCs) do not express a unique definite epitope or marker gene. As such, minimal criteria were recently established for defining multipotent MSC. These criteria include expression of CD73, CD90, CD105, and a lack of hematopoietic marker expression. However, we detected binding of a CD14 antibody on bone marrow- and placenta-derived MSC and investigated the staining of CD14 antibodies on these MSC in more detail. The MSC were isolated from human bone marrow and placenta tissue, expanded, characterized by quantitative RT-PCR, flow cytometry, and immunocytochemistry and differentiated to generate osteoblasts, chondrocytes, and adipocytes. The CD14-cross-reactive MSCs were enriched by cell sorting. Human peripheral blood mononuclear cells, fibroblasts, and hematopoietic cell lines served as controls. Utilizing four different clones of CD14 monoclonal antibodies, we found that three CD14 reagents stained the MSC. Two CD14 antibodies (HCD14 and M5E2) clearly marked the CD90(+) MSC population with distinct intensities, clone 134 620 generated a shift in flow cytometry histograms, but clone MPhiP9 did not stain MSC. Transcripts encoding CD14 or the CD14 protein were not detected in MSC. We confirm that bone marrow- and placenta-derived MSC do not express CD14 and that the CD14 antibody MPhiP9 discriminates between monocytes and MSC more efficiently than the other antibodies employed here. This investigation does not contradict previous work but provides a more accurate characterization of MSC.
Abstract: Inhibition of recombinant D-amino acid oxidase from Trigonopsis variabilis (TvDAAO) activity in the presence of different sodium salts and potassium chloride is reported. A competitive inhibition pattern by sodium chloride was observed, and an inhibition constant value of K(i) = 85 mM was calculated. Direct connection of NaCl inhibition with FAD cofactor dissociation was confirmed by measuring the fluorescence of tryptophanyl residues of the holoenzyme.
Abstract: BACKGROUND: Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3)-induced myocarditis. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR) and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-gamma. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-gamma mRNA expression. CONCLUSIONS: We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis.
Abstract: During secondary fracture healing, various tissue types including new bone are formed. The local mechanical strains play an important role in tissue proliferation and differentiation. To further our mechanobiological understanding of fracture healing, a precise assessment of local strains is mandatory. Until now, static analyses using Finite Elements (FE) have assumed homogenous material properties. With the recent quantification of both the spatial tissue patterns (Vetter et al., 2010) and the development of elastic modulus of newly formed bone during healing (Manjubala et al., 2009), it is now possible to incorporate this heterogeneity. Therefore, the aim of this study is to investigate the effect of this heterogeneity on the strain patterns at six successive healing stages. The input data of the present work stemmed from a comprehensive cross-sectional study of sheep with a tibial osteotomy (Epari et al., 2006). In our FE model, each element containing bone was described by a bulk elastic modulus, which depended on both the local area fraction and the local elastic modulus of the bone material. The obtained strains were compared with the results of hypothetical FE models assuming homogeneous material properties. The differences in the spatial distributions of the strains between the heterogeneous and homogeneous FE models were interpreted using a current mechanobiological theory (Isakson et al., 2006). This interpretation showed that considering the heterogeneity of the hard callus is most important at the intermediate stages of healing, when cartilage transforms to bone via endochondral ossification.
Abstract: At its highest level of microstructural organization-the mesoscale or millimeter scale-cortical bone exhibits a heterogeneous distribution of pores (Haversian canals, resorption cavities). Multi-scale mechanical models rely on the definition of a representative volume element (RVE). Analytical homogenization techniques are usually based on an idealized RVE microstructure, while finite element homogenization using high-resolution images is based on a realistic RVE of finite size. The objective of this paper was to quantify the size and content of possible cortical bone mesoscale RVEs. RVE size was defined as the minimum size: (1) for which the apparent (homogenized) stiffness tensor becomes independent of the applied boundary conditions or (2) for which the variance of elastic properties for a set of microstructure realizations is sufficiently small. The field of elastic coefficients and microstructure in RVEs was derived from one acoustic microscopy image of a human femur cortical bone sample with an overall porosity of 8.5%. The homogenized properties of RVEs were computed with a finite element technique. It was found that the size of the RVE representative of the overall tissue is about 1.5 mm. Smaller RVEs (~0.5 mm) can also be considered to estimate local mesoscopic properties that strongly depend on the local pores volume fraction. This result provides a sound basis for the application of homogenization techniques to model the heterogeneity of cortical microstructures. An application of the findings to estimate elastic properties in the case of a porosity gradient is briefly presented.
Abstract: Background: Bone morphogenetic proteins (BMPs) are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway. xD;Methodology/Principal Findings: Here we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway) and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP) production and qPCR analysis of osteoblast marker gene expression. xD;Conclusions/Significance: Our study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an additional mechanism for the cell to respond to BMP in a context specific manner. Moreover, we suggest a novel Smad dependent signal cascade induced by BMP-2, which does not require endocytosis.
Abstract: The trace element selenium is an essential micronutrient that is required for the biosynthesis of selenocysteine-containing selenoproteins. Most of the known selenoproteins are expressed in the thyroid gland, including some with still unknown functions. Among the well-characterized selenoproteins are the iodothyronine deiodinases, glutathione peroxidases and thioredoxin reductases, enzymes involved in thyroid hormone metabolism, regulation of redox state and protection from oxidative damage. Selenium content in selenium-sensitive tissues such as the liver, kidney or muscle and expression of nonessential selenoproteins, such as the glutathione peroxidases GPx1 and GPx3, is controlled by nutritional supply. The thyroid gland is, however, largely independent from dietary selenium intake and thyroid selenoproteins are preferentially expressed. As a consequence, no explicit effects on thyroid hormone profiles are observed in healthy individuals undergoing selenium supplementation. However, low selenium status correlates with risk of goiter and multiple nodules in European women. Some clinical studies have demonstrated that selenium-deficient patients with autoimmune thyroid disease benefit from selenium supplementation, although the data are conflicting and many parameters must still be defined. The baseline selenium status of an individual could constitute the most important parameter modifying the outcome of selenium supplementation, which might primarily disrupt self-amplifying cycles of the endocrine-immune system interface rectifying the interaction of lymphocytes with thyroid autoantigens. Selenium deficiency is likely to constitute a risk factor for a feedforward derangement of the immune system-thyroid interaction, while selenium supplementation appears to dampen the self-amplifying nature of this derailed interaction.
Abstract: Um Frakturheilungszeiten zu minimieren und Patienten eine möglichst schnelle und weitestgehende Rehabilitation zu ermöglichen, ist ein Verständnis sowohl der biologischen Prozesse der Heilung als auch der mechanischen Bedingungen, unter denen diese Heilung vonstatten geht, nötig. Wissen um die mechanischen Bedingungen ist insbesondere deshalb von Bedeutung, da die biologischen Prozesse der Heilung und Regeneration von den durch Muskel- und Gelenkkräfte definierten Rahmenbedingungen wesentlich beeinflusst werden. Durch die Wahl der Osteosynthese können die mechanischen Rahmenbedingungen an der Fraktur und dem physikalischen Stimulus, dem die zellulären Strukturen ausgesetzt sind, weiter moduliert werden. Unabhängig von der Methode der Frakturstabilisierung ist die sekundäre Knochenheilung immer einer komplexen interfragmentären Bewegung ausgesetzt. Dabei beeinflussen die auftretenden Fragmentbewegungen den Heilungsprozess in seiner Art und Schnelligkeit.
Abstract: Myocardial infarction is associated with persistent muscle damage, scar formation and depressed cardiac performance. Recent studies have demonstrated the clinical significance of stem cell-based therapies after myocardial infarction with the aim to improve cardiac remodeling and function by inducing the reconstitution of functional myocardium and formation of new blood vessels. Stem cell homing signals play an important role in stem cell mobilization from the bone marrow to the ischemic cardiac environment and are therefore crucial for myocardial repair. To date, the most prominent stem cell homing factor is the chemokine SDF-1[alpha]/CXCL12. This protein was shown to be significantly upregulated in many experimental models of myocardial infarction and in patients suffering from ischemic cardiac diseases, suggesting the involvement in the pathophysiology of these disorders. A number of studies focused on manipulating SDF-1[alpha] and its receptor CXCR4 as central regulators of the stem cell mobilization process. Targeted expression of SDF-1[alpha] after myocardial infarction was shown to result in increased engraftment of bone marrow-derived stem cells into infarcted myocardium. This was accompanied by beneficial effects on cardiomyocyte survival, neovascularization and cardiac function. Thus, the SDF-1/CXCR4 axis seems to be a promising novel therapeutic approach to improve post-infarction therapy by attracting circulating stem cells to remain, survive and possibly differentiate in the infarct area. This review will summarize clinical trials of stem cell therapy in patients with myocardial infarction. We further discuss the basic findings about SDF-1[alpha] in stem cell recruitment and its therapeutic implications in experimental myocardial infarction.
Abstract: The determination of an accurate centre of rotation (CoR) from skin markers is essential for the assessment of abnormal gait patterns in clinical gait analysis. Despite the many functional approaches to estimate CoRs, no non-invasive analytical determination of the error in the reconstructed joint location is currently available. The purpose of this study was therefore to verify the residual of the symmetrical centre of rotation estimation (SCoRE) as a reliable indirect measure of the error of the computed joint centre. To evaluate the SCoRE residual, numerical simulations were performed to evaluate CoR estimations at different ranges of joint motion. A statistical model was developed and used to determine the theoretical relationships among the SCoRE residual, the magnitude of the skin marker artefact, the corrections to the marker positions, and the error of the CoR estimations to the known centre of rotation. We found that the equation err=0.5rs provides a reliable relationship among the CoR error, err, and the scaled SCoRE residual, rs, providing that any skin marker artefact is first minimised using the optimal common shape technique (OCST). Measurements on six healthy volunteers showed a reduction of SCoRE residual from 11 to below 6Â mm and therefore demonstrated consistency of the theoretical considerations and numerical simulations with the in vivo data. This study also demonstrates the significant benefit of the OCST for reducing skin marker artefact and thus for predicting the accuracy of determining joint centre positions in functional gait analysis. For the first time, this understanding of the SCoRE residual allows a measure of error in the non-invasive assessment of joint centres. This measure now enables a rapid assessment of the accuracy of the CoR as well as an estimation of the reproducibility and repeatability of skeletal motion patterns.
Abstract: Adiponectin (APN), a cytokine constitutively produced in fat tissue, has been shown to exert anti-inflammatory effects in various disease models. While the influence of APN on monocytic cells has been extensively studied in vitro, little is known about its role in T cells. In this study, we show that while <10% of human peripheral blood T cells express adiponectin receptors (AdipoRs) on their surface, most T cells store AdipoRs in intracellular compartments. AdipoRs colocalized with immune regulatory molecules CTLA-4 and TIRC7 within clathrin-coated vesicles. After stimulation, the expression of adiponectin receptor 1 (AdipoR1) and AdipoR2 was upregulated on the surface of antigen-specific T cells, as determined by tetramer or CD137 staining, and AdipoR1 and AdipoR2 coexpressed with CTLA-4. Addition of APN resulted in a significant diminution of antigen-specific T-cell expansion. Mechanistically, APN enhanced apoptosis and inhibited proliferation of antigen-specific T-cell lines. Further, APN directly inhibited cytokine production in response to antigen stimulation. In line with the in vitro data, APN-deficient (knockout, KO) mice had higher frequencies of CD137+ T cells upon Coxsackie B virus infection. Altogether, our data suggest that APN is a novel negative T-cell regulator. In contrast to the CTLA-4 ligand B7 only expressed on APCs, APN is abundant in human plasma.
Abstract: Background and purpose: Animal models of skeletal muscle injury should be thoroughly described and should mimic the clinical situation. We established a model of a critical size crush injury of the soleus muscle in rats. The aim was to describe the time course of skeletal muscle regeneration using mechanical, histological, and magnetic resonance (MR) tomographic methods. xD;Methods: Left soleus muscles of 36 Sprague-Dawley rats were crushed in situ in a standardized manner. We scanned the lower legs of 6 animals by 7-tesla MR one week, 4 weeks, and 8 weeks after trauma. Regeneration was evaluated at these times by in vivo measurement of muscle contraction forces after fast-twitch and tetanic stimulation (groups 1W, 4W, 8W; 6 per group). Histological and immunohistological analysis was performed and the amount of fibrosis within the injured muscles was determined histomorphologically. xD;Results: MR signals of the traumatized soleus muscles showed a clear time course concerning microstructure and T1 and T2 signal intensity. Newly developed neural endplates and myotendinous junctions could be seen in the injured zones of the soleus. Tetanic force increased continuously, starting at 23% (SD 4) of the control side (p < 0.001) 1 week after trauma and recovering to 55% (SD 23) after 8 weeks. Fibrotic tissue occupied 40% (SD 4) of the traumatized muscles after the first week, decreased to approximately 25% after 4 weeks, and remained at this value until 8 weeks. xD;Interpretation: At both the functional level and the morphological level, skeletal muscle regeneration follows a distinct time course. Our trauma model allows investigation of muscle regeneration after a standardized injury to muscle fibers.
Abstract: Mechanobiological theories have been introduced to illustrate the interaction between biology and the local mechanical environment during bone healing. Although several theories have been proposed, a quantitative validation using histomorphometric data is still missing. In this study, in vivo histological data based on an ovine animal experiment was quantified and used to validate bone healing simulations focussing on the endochondral ossification process. The bone formation at different callus regions (periosteal and endosteal bone at the medial and lateral side) was analyzed from in vivo data and quantitatively compared with in silico results. A histomorphometric difference was found in medial and lateral hard callus formation 3 weeks after osteotomy. However, the same amount of new bone was formed on both sides between week 3 and 6. Using a parametric approach, distinct ranges for mechanical strain levels regulating tissue formation were found, for which the in silico prediction agrees with the in vivo endochondral ossification both in pattern and quantity. According to this finding, a strain range of 1 to 8% seems to be conducive for cartilage formation while bone formation may be facilitated by strains up to 4%. This study demonstrates the potential of a thorough validation of in silico results for gaining a better understanding of mechanobiological mechanisms during bone healing.
Notes: Witt, Florian xD;Petersen, Ansgar xD;Seidel, Robin xD;Vetter, Andreas xD;Weinkamer, Richard xD;Duda, Georg N xD;Ann Biomed Eng. 2011 Oct;39(10):2531-41. Epub 2011 Jun 21.
Abstract: Hypoxia, a feature of inflammation and tumors, is a potent inducer of the proinflammatory cytokine macrophage migration inhibitory factor (MIF). In transformed cells, MIF was shown to modulate and to be modulated via the oxygen-sensitive transcription factor hypoxia-inducible factor (HIF)-1. Furthermore, anti-inflammatory glucocorticoids (GCs) were described to regulate MIF action. However, in-depth studies of the interaction between MIF and HIF-1 and GC action in nontransformed primary human CD4+ T cells under hypoxia are missing. Therefore, we investigated the functional relationship between MIF and HIF and the impact of the GC dexamethasone (DEX) on these key players of inflammation in human CD4+ T cells. In this article, we show that hypoxia, and specifically HIF-1, is a potent and rapid inducer of MIF expression in primary human CD4+ T cells, as well as in Jurkat T cells. MIF signaling via CD74, in turn, is essential for hypoxia-mediated HIF-1α expression and HIF-1 target gene induction involving ERK/mammalian target of rapamycin activity complemented by PI3K activation upon mitogen stimulation. Furthermore, MIF signaling enhances T cell proliferation under normoxia but not hypoxia. MIF also counterregulates DEX-mediated suppression of MIF and HIF-1α expression. Based on these data, we suggest that hypoxia significantly affects the expression of HIF-1α in a MIF-dependent manner leading to a positive-feedback loop in primary human CD4+ T cells, thus influencing the lymphoproliferative response and DEX action via the GC receptor. Therefore, we suggest that HIF and/or MIF could be useful targets to optimize GC therapy when treating inflammation.
Abstract: We investigated the accuracy of implant positioning during total hip arthroplasty (THA) through a minimally invasive approach in relation to body mass index (BMI) and gender by assessing 48 patients. Functional and radiological parameters were evaluated. BMI positively correlated with operation time (p=0.04), but BMI and gender had no influence on implant positioning. The Harris hip score (HHS) increased significantly (46.5 +/- 11.8 preoperatively, 92.1 +/- 9.7 postoperatively, p<0.0001). The surgical approach described resulted in reproducibility of implant positioning independent of influence by BMI or gender.
Abstract: ABSTRACT: BACKGROUND: Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naive T cells as possible key players in immune regulation directly in the CNS is still highly debated. METHODS: We applied ex vivo and intravital TPLSM to investigate migratory pathways of naive T cells in the inflamed and non-inflamed CNS. MACS-sorted naive CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures. RESULTS: By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naive T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naive CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naive T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naive CD4+ T cell tracks. CONCLUSIONS: The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naive CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naive CD4+ T cells within the diseased CNS.
Abstract: Background: Sleep disorders can cause tiredness. The relationship between sleep disorders and fatigue in patients with multiple sclerosis (MS) has not yet been investigated systematically. Objective: To investigate the relationship between fatigue and sleep disorders in patients with MS. Methods: Some 66 MS patients 20 to 66 years old were studied by overnight polysomnography. Using a cut-off point of 45 in the Modified Fatigue Impact Scale (MFIS), the entire cohort was stratified into a fatigued MS subgroup (n = 26) and a non-fatigued MS subgroup (n = 40). Results: Of the fatigued MS patients, 96% (n = 25) were suffering from a relevant sleep disorder, along with 60% of the non-fatigued MS patients (n = 24) (p = 0.001). Sleep-related breathing disorders were more frequent in the fatigued MS patients (27%) than in the non-fatigued MS patients (2.5%). Significantly higher MFIS values were detected in all (fatigued and non-fatigued) patients with relevant sleep disorders (mean MFIS 42.8; SD 18.3) than in patients without relevant sleep disorders (mean MFIS 20.5; SD 17.0) (p < 0.001). Suffering from a sleep disorder was associated with an increased risk of fatigue in MS (odds ratio: 18.5; 95% CI 1.6–208; p = 0.018). Conclusion: Our results demonstrate a clear and significant relationship between fatigue and sleep disorders.
Abstract: Bone is a dynamic tissue that is continually undergoing a process of remodeling - an effect due to the interplay between bone resorption by osteoclasts and bone formation by osteoblasts. When new bone is deposited, some of the osteoblasts are embedded in the mineralizing collagen matrix and differentiate to osteocytes, forming a dense network throughout the whole bone tissue. Here, we investigate the extent to which the organization of the osteocyte network controls the collagen matrix arrangement found in various bone tissues. Several tissue types from equine, ovine and murine bone have been examined using confocal laser scanning microscopy as well as polarized light microscopy and back-scattered electron imaging. From comparing the spatial arrangements of unorganized and organized bone, we propose that the formation of a highly oriented collagen matrix requires an alignment of osteoblasts whereby a substrate layer provides a surface such that osteoblasts can align and, collectively, build new matrix. Without such a substrate, osteoblasts act isolated and only form matrices without long range order. Hence, we conclude that osteoblasts synthesize and utilize scaffold-like primary tissue as a guide for the deposition of highly ordered and mechanically competent bone tissue by a collective action of many cells.
Abstract: Ultrasound is regarded as a promising method to determine the intramuscular fat content of pork loin. At intact carcasses, the signal passes the backfat whose ultrasound parameters (sound velocity and attenuation) have not been fully investigated. This study intended to collect a dataset of ultrasound parameters for individual backfat layers and to elucidate relationships with structural and compositional characteristics. In-vitro measurements at 10 MHz were conducted on backfat samples of pork carcasses representative for German populations. The average sound velocity ranged from 1436 ± 9 to 1470 ± 37 ms- 1 for the fat layers, and 1682 ± 23 ms- 1 for skin. Velocity of the compound backfat decreased with overall thickness. Attenuation was not affected by thickness ranging between 1.6 ± 0.7 and 2.7 ± 1.5 dB MHz- 1 cm- 1 for all layers. Sound velocity was negatively correlated with fat content and dry matter. The obtained results are anticipated to improve signal correction prior to spectral analysis of ultrasound measurements at intact carcasses.
Abstract: Ultrasound velocity and attenuation of soft tissues have been widely investigated. However, few studies completely covered considerable variations of both, structure and composition. The aim of this study was to collect acoustic reference data of porcine Longissimus muscle and associate them with compositional traits. In addition, measurements were conducted on fresh, formalin fixed, and frozen-thawed samples to evaluate the effect of processing on ultrasound parameters and comparisons with earlier investigations. Measurement conditions (temperature and fibre orientation) were realised close to hanging carcasses conditions. Sound velocity ranged from 1617 ± 6 to 1622 ± 5 ms-1, while attenuation mostly ranged from 1.0 ± 0.3 to 1.2 ± 0.3 dB MHz-1 cm-1. Only formalin fixed samples showed significantly higher attenuation (2.2 ± 0.6 dB MHz-1 cm-1). Highest correlations have been observed between intramuscular fat and attenuation (up to r = .7). The obtained results are anticipated to improve ultrasound based estimation of the intramuscular fat of pig muscle on intact carcasses.
Abstract: This paper reviews recent literature about the physical processes involved in cell interactions and tissue development. Rather than being exhaustive, we intend to provide illustrative examples of experiments and theoretical approaches into how cells interact with other cells and with substrates to form complex tissues and organs. Forces and geometry efficiently coordinate cell behaviour through feedback and mechanical homeostasis, leading to emergent properties not directly evident from the behaviour of individual cells. Two important examples for such emergent properties are the patterning of growth and differentiation within tissues, and the long-range organisation of the extracellular matrix. Despite the complexity of the biological, chemical and mechanical processes involved, theoretical studies have shown that many of these phenomena can be described quantitatively by simple physical processes, such as surface tension controlled growth. In addition to improving knowledge about the biology of tissues, a thorough theoretical understanding of the self-organising mechanisms used by nature may provide inspiration for the design of self-assembling biomimetic soft materials.
Abstract: The mechanical properties of bone are known to depend on its structure at all length scales. In large animals, such as sheep, cortical bone grows very quickly and it is known that this occurs in 2 stages whereby a poorly ordered (mostly woven) bone structure is initially deposited and later augmented and partially replaced by parallel fibered and lamellar bone with much improved mechanical properties, often called primary osteons. Most interestingly, a similar sequence of events has also recently been observed during callus formation in a sheep osteotomy model. This has prompted the idea that fast intramembranous bone formation requires an intermediate step where bone with a lower degree of collagen orientation is deposited first as a substrate for osteoblasts to coordinate the synthesis of lamellar tissue. Since some osteoblasts become embedded in the mineralizing collagen matrix which they synthesize, the resulting osteocyte network is a direct image of the location of osteoblasts during bone formation. Using 3-dimensional imaging of osteocyte networks as well as tissue characterization by polarized light microscopy and backscattered electron imaging, we revisit the structure of growing plexiform (fibrolamellar) bone and callus in sheep. We show that bone deposited initially is based on osteocytes without spatial correlation and encased in poorly ordered matrix. Bone deposited on top of this has lamellar collagen orientation as well as a layered arrangement of osteocytes, both parallel to the surfaces of the initial tissue. This supports the hypothesis that the initial bone constitutes an endogenous scaffold for the subsequent deposition of parallel fibered and lamellar bone.
Abstract: BACKGROUND: BKV-associated nephropathy represents a serious complication of the posttransplant period in kidney transplant recipients. Monitoring BKV-specific immunity is of a special importance for estimation of clinical course in patients with BKV reactivation. Our recent data demonstrated that all five BKV antigens are immunogenic and elicit T-cell responses varying within patients. Therefore, all five BKV proteins should be evaluated for the assessment of BKV-specific immunity. However, analysis of five proteins performed separately is time- and cost-intensive and requires large amount of blood. METHODS: Using novel approach of a mixture of overlapping peptide pools encompassing all five BKV antigens (viral protein [VP] 1, VP2, VP3, large tumor antigen, and small tumor antigen) and multiparameter flow cytometry, we evaluate BKV-specific T cells in patients with a previous/present severe long-lasting or transient BKV reactivation. Patients without BKV reactivation were used as control. RESULTS: In this study, we show that using mixture of overlapping peptide pool results in the magnitude of CD4- and CD8-positive BKV-specific T-cell response, which is significantly higher compared with any frequencies detected by previously used single BKV antigen stimulation. Of interest, patients with a history of rapid BKV clearance had significantly higher frequency of multifunctional interferon gamma-gamma/interleukin (IL)-2/tumor necrosis factor-alpha and IL-2/tumor necrosis factor-alpha CD4-positive T cells, suggesting protective potential of polyfunctional T cells. Furthermore, we did not find IL-17-producing BKV-specific memory T cells in patients recovered from BKV reactivation. CONCLUSIONS: Here, we established a fast and sensitive approach allowing the most comprehensive assessment of the total BKV immunity performed to date and offer a new platform for further prospective studies.
Abstract: Summary IL-6 and IL-10 have previously been implicated in the pathogenesis of post-transplant lymphoproliferative disorders (PTLD) and, like peripheral lymphocyte populations, are markers of immune status that are amenable to study in vivo. Thus, we analyzed cytokine plasma levels as well as lymphocyte subsets in a longitudinal analysis of 38 adult transplant recipients undergoing treatment for PTLD. Pretherapeutically, we found significantly elevated IL-6 (13.8 pg/ml) and IL-10 plasma levels (54.7 pg/ml) – in the case of IL-10, even higher in treatment nonresponders than in responders (116 vs. 14 pg/ml). Over time, however, IL-10 levels did not correlate with the course of disease, whereas those of IL-6 did, falling in responders and rising in nonresponders. These findings were independent of histological EBV-status, treatment type, and total peripheral T-cell counts, which were significantly reduced in patients with PTLD. Our observations support the idea that although IL-10 is important for creating a permissive environment for post-transplant lymphoma development, IL-6 is associated with PTLD proliferation. The analysis of lymphocyte subsets further identified HLA-DR+ CD8+ lymphocyte numbers as significantly different in non-PTLD controls (33%), treatment responders (44%) and nonresponders (70%). Although the specificity of these cells is unclear, their increase might correlate with the impaired tumor-specific cytotoxic-T-lymphocyte (CTL)-response in PTLD.
Abstract: The protective effects of high-density lipoprotein (HDL) under lipopolysaccharide (LPS) conditions have been well documented. Here, we investigated whether an effect of HDL on Toll-like receptor 4 (TLR4) expression and signalling may contribute to its endothelial-protective effects and to improved survival in a mouse model of LPS-induced inflammation and lethality. HDL cholesterol increased 1.7-fold (p<0.005) and lung endothelial TLR4 expression decreased 8.4-fold (p<0.005) 2 weeks after apolipoprotein (apo) A-I gene transfer. Following LPS administration in apo A-I gene transfer mice, lung TLR4 and lung MyD88 mRNA expression, reflecting TLR4 signalling, were 3.0-fold (p<0.05) and 2.1-fold (p<0.05) lower, respectively, than in LPS control mice. Concomitantly, LPS-induced lung neutrophil infiltration, lung oedema and mortality were significantly attenuated following apo A-I transfer. In vitro, supplementation of HDL or apo A-I to human microvascular endothelial cells-1 24 h before LPS administration reduced TLR4 expression, as assessed by fluorescent-activated cell sorting, and decreased the LPS-induced MyD88 mRNA expression and NF-kappaB activity, independently of LPS binding. In conclusion, HDL reduces TLR4 expression and signalling in endothelial cells, which may contribute significantly to the protective effects of HDL in LPS-induced inflammation and lethality.
Abstract: BACKGROUND: Selenium (Se) status in non-deficient subjects is typically assessed by the Se contents of plasma/serum. That pool comprises two functional, specific selenoprotein components and at least one non-functional, non-specific components which respond differently to changes in Se intake. A more informative means of characterizing Se status in non-deficient individuals is needed. METHODS: Multiple biomarkers of Se status (plasma Se, serum selenoprotein P [SEPP1], plasma glutathione peroxidase activity [GPX3], buccal cell Se, urinary Se) were evaluated in relation to selenoprotein genotypes (GPX1, GPX3, SEPP1, SEP15), dietary Se intake, and parameters of single-carbon metabolism in a cohort of healthy, non-Se-deficient men (n = 106) and women (n = 155). CONCLUSIONS: Plasma Se concentration was 142.0 +/- 23.5 ng/ml, with GPX3 and serum-derived SEPP1 calculated to comprise 20% and 34%, respectively, of that total. The balance, comprised of non-specific components, accounted for virtually all of the interindividual variation in total plasma Se. Buccal cell Se was associated with age and plasma homocysteine (hCys), but not plasma Se. SEPP1 showed a quadratic relationship with body mass index, peaking at BMI 25-30. Urinary Se was greater in women than men, and was associated with metabolic body weight (kg0.75), plasma folate, vitamin B12 and hCys (negatively). One GPX1 genotype (679T/T) was associated with significantly lower plasma Se levels than other allelic variants. Selenium intake, estimated from food frequency questionnaires, did not predict Se status as indicated by any biomarker. These results show that genotype, methyl-group status and BMI contribute to variation in Se biomarkers in Se-adequate individuals.
Abstract: Inflammatory cardiomyopathy is associated with a diffuse inflammation in the heart accompanied with cardiac fibrosis, cardiomyocyte apoptosis, and reduced capillary density. On the other hand, mesenchymal stromal cells (MSCs) have immunomodulatory, anti-fibrotic, anti-apoptotic, and pro-angiogenic features, making them attractive candidates for the treatment of inflammatory cardiomyopathy. The potential of MSC application for the treatment of myocarditis is supported by their beneficial effects in experimental models of acute and chronic inflammatory cardiomopathy. This review summarizes the cardioprotective features of MSCs and describes how MSCs are primed by the inflammatory environment to exert their protective effects. In view of clinical translation, searching for the optimal source of MSC and delivery route, allowing efficient and non-invasive cell application, the differences between MSCs of distinct origin and between diverse routes of application are outlined.
Abstract: AIMS: Coxsackievirus B3 (CVB3)-induced myocarditis, initially considered a sole immune-mediated disease, also results from a direct CVB3-mediated injury of the cardiomyocytes. Mesenchymal stem cells (MSCs) have, besides immunomodulatory, also anti-apoptotic features. In view of clinical translation, we first analysed whether MSCs can be infected by CVB3. Next, we explored whether and how MSCs could reduce the direct CVB3-mediated cardiomyocyte injury and viral progeny release, in vitro, in the absence of immune cells. Finally, we investigated whether MSC application could improve murine acute CVB3-induced myocarditis. METHODS AND RESULTS: Phase contrast pictures and MTS viability assay demonstrated that MSCs did not suffer from CVB3 infection 4-12-24-48 h after CVB3 infection. Coxsackievirus B3 RNA copy number decreased in this time frame, suggesting that no CVB3 replication took place. Co-culture of MSCs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis, oxidative stress, intracellular viral particle production, and viral progeny release in a nitric oxide (NO)-dependent manner. Moreover, MSCs required priming via interferon-gamma (IFN-gamma) to exert their protective effects. In vivo, MSC application improved the contractility and relaxation parameters in CVB3-induced myocarditis, which was paralleled with a reduction in cardiac apoptosis, cardiomyocyte damage, left ventricular tumour necrosis factor-alpha mRNA expression, and cardiac mononuclear cell activation. Mesenchymal stem cells reduced the CVB3-induced CD4- and CD8- T cell activation in an NO-dependent way and required IFN-gamma priming. CONCLUSION: We conclude that MSCs improve murine acute CVB3-induced myocarditis via their anti-apoptotic and immunomodulatory properties, which occur in an NO-dependent manner and require priming via IFN-gamma.
Abstract: Dishevelled (Dv1) is a multifunctional effector of different Wnt cascades. Both canonical Wnt3a and noncanonical Wnt5a stimulate casein-kinase-1 (CK1) -mediated phosphorylation of Dv1, visualized as electrophoretic mobility shift [phosphorylated and shifted Dv1 (ps-Dv1)]. However, the role of this phosphorylation remains obscure. Here we report the functional interaction of ps-Dv1 with the receptor tyrosine kinase Ror2, which is an alternative Wnt receptor and is able to inhibit canonical Wnt signaling. We demonstrate interaction between Ror2 and ps-Dv1 at the cell membrane after Wnt3a or Wnt5a stimulus dependent on CK1. Ps-Dv1 interacts with the C-terminal proline-serine-threonine-rich domain of Ror2, which is required for efficient inhibition of canonical Wnt signaling. We further show that the Dv1 C terminus, which seems to be exposed in ps-Dv1 and efficiently binds Ror2, is an intrinsic negative regulator of the canonical Wnt pathway downstream of beta-catenin. The Dv1 C terminus is necessary and sufficient to inhibit canonical Wnt/beta-catenin signaling, which is dependent on the presence of Ror2. Furthermore, both the Dv1 C terminus and CK1 epsilon can inhibit the Wnt5a/Ror2/ATF2 pathway in mammalian cells and Xenopus explant cultures. This suggests that phosphorylation of Dv1 triggers negative feedback regulation for different branches of Wnt signaling in a Ror2-dependent manner.-Witte, F., Bernatik, O., Kirchner, K., Masek, J., Mahl, A., Krejci, P., Mundlos, S., Schambony, A., Bryja, V., Stricker, S. Negative regulation of Wnt signaling mediated by CK1-phosphorylated Dishevelled via Ror2. FASEB J. 24, 2417-2426 (2010). www.fasebj.org
Abstract: Summary Neuronal damage in autoimmune neuroinflammation is the correlate for long-term disability in multiple sclerosis (MS) patients. Here, we investigated the role of immune cells in neuronal damage processes in animal models of MS by monitoring experimental autoimmune encephalomyelitis (EAE) by using two-photon microscopy of living anaesthetized mice. In the brainstem, we detected sustained interaction between immune and neuronal cells, particularly during disease peak. Direct interaction of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 and neuronal cells in demyelinating lesions was associated with extensive axonal damage. By combining confocal, electron, and intravital microscopy, we showed that these contacts remarkably resembled immune synapses or kinapses, albeit with the absence of potential TÂ cell receptor engagement. Th17 cells induced severe, localized, and partially reversible fluctuation in neuronal intracellular Ca2+ concentration as an early sign of neuronal damage. These results highlight the central role of the Th17 cell effector phenotype for neuronal dysfunction in chronic neuroinflammation.
Abstract: Human amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnostics procedures. Recently, it has been illustrated that these cells may also serve as a valuable model system to study developmental processes and for application in regenerative therapies. Cellular reprogramming is a means of assigning greater value to primary AFCs by inducing self-renewal and pluripotency and, thus, bypassing senescence. Here, we report the generation and characterization of human amniotic fluid-derived induced pluripotent stem cells (AFiPSCs) and demonstrate their ability to differentiate into the trophoblast lineage after stimulation with BMP2/BMP4. We further carried out comparative transcriptome analyses of primary human AFCs, AFiPSCs, fibroblast-derived iPSCs (FiPSCs) and embryonic stem cells (ESCs). This revealed that the expression of key senescence-associated genes are down-regulated upon the induction of pluripotency in primary AFCs (AFiPSCs). By defining distinct and overlapping gene expression patterns and deriving the LARGE (Lost, Acquired and Retained Gene Expression) Principle of Cellular Reprogramming, we could further highlight that AFiPSCs, FiPSCs and ESCs share a core self-renewal gene regulatory network driven by OCT4, SOX2 and NANOG. Nevertheless, these cell types are marked by distinct gene expression signatures. For example, expression of the transcription factors, SIX6, EGR2, PKNOX2, HOXD4, HOXD10, DLX5 and RAXL1, known to regulate developmental processes, are retained in AFiPSCs and FiPSCs. Surprisingly, expression of the self-renewal-associated gene PRDM14 or the developmental processes-regulating genes WNT3A and GSC are restricted to ESCs. Implications of this, with respect to the stability of the undifferentiated state and long-term differentiation potential of iPSCs, warrant further studies.
Abstract: After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor–mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell–mediated immune responses and open new therapeutic possibilities.
Abstract: Marker-based gait analysis of the lower limb that uses assumptions of generic anatomical morphology can be susceptible to errors, particularly in subjects with high levels of soft tissue coverage. We hypothesize that a functional approach for assessing skeletal kinematics, based on the application of techniques to reduce soft tissue artefact and functionally identify joint centres and axes, can more reliably (repeatably and reproducibly) assess the skeletal kinematics than a standard generic regression approach. Six healthy adults each performed 100 repetitions of a standardized motion, measured on four different days and by five different observers. Using OSSCA, a combination of functional approaches to reduce soft tissue artefact and identify joint centres and axes, the lengths of the femora and tibiae were determined to assess the inter-day and inter-observer reliability, and compared against a standard generic regression approach. The results indicate that the OSSCA was repeatable and reproducible (ICC lowest bound 0.87), but also provided an improvement over the regression approach (ICC lowest bound 0.69). Furthermore, the analysis of variance revealed a statistically significant variance for the factor "observers" (p < 0.01; low-reproducibility) when using the regression approach for determining the femoral lengths. Here, this non-invasive, rapid and robust approach has been demonstrated to allow the repeatable and reproducible identification of skeletal landmarks, which is insensitive to marker placement and measurement session. The reliability of the OSSCA thus allows its application in clinical studies for reducing the uncertainty of approach-induced systematic errors.
Abstract: Background: Colorectal cancer (CRC) is with approximately 1 million cases the third most common cancer worldwide. Extensive research is ongoing to decipher the underlying genetic patterns with the hope to improve early cancer diagnosis and treatment. In this direction, the recent progress in next generation sequencing technologies has revolutionized the field of cancer genomics. However, one caveat of these studies remains the large amount of genetic variations identified and their interpretation. xD;Methodology/Principal Findings: Here we present the first work on whole exome NGS of primary colon cancers. We performed 454 whole exome pyrosequencing of tumor as well as adjacent not affected normal colonic tissue from microsatellite stable (MSS) and microsatellite instable (MSI) colon cancer patients and identified more than 50,000 small nucleotide variations for each tissue. According to predictions based on MSS and MSI pathomechanisms we identified eight times more somatic non-synonymous variations in MSI cancers than in MSS and we were able to reproduce the result in four additional CRCs. Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions. In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function. xD;Conclusions/Significance: We conclude that with deep sequencing of tumor exomes one may be able to predict the microsatellite status of CRC and in addition identify potentially clinically relevant mutations.
Abstract: Attention is one of the cognitive domains typically affected in multiple sclerosis. The Attention Network Test was developed to measure the function of the three distinct attentional networks, alerting, orienting, and executive control. The Attention Network Test has been performed in various neuropsychiatric conditions, but not in multiple sclerosis. Our objective was to investigate functions of attentional networks in multiple sclerosis by means of the Attention Network Test. Patients with relapsing—remitting multiple sclerosis (n = 57) and healthy controls (n = 57) matched for age, sex, and education performed the Attention Network Test. Significant differences between patients and controls were detected in the alerting network (p = 0.003), in contrast to the orienting (p = 0.696) and the conflict (p = 0.114) network of visual attention. Mean reaction time in the Attention Network Test was significantly longer in multiple sclerosis patients than in controls (p = 0.032), Multiple sclerosis patients benefited less from alerting cues for conflict resolution compared with healthy controls. The Attention Network Test revealed specific alterations of the attention network in multiple sclerosis patients which were not explained by an overall cognitive slowing.
Abstract: Selenium modifies inflammatory reactions in rodents and humans. The liver controls metabolism and transport of selenium via hepatically-derived SEPP (selenoprotein P). Intracellular SEPS (selenoprotein S) modifies endoplasmic-reticulum function and immune-cell activity. Polymorphisms in SEPS have been associated with cytokine levels and inflammatory diseases in a subset of clinical studies. In the present study, we hypothesized that sex and selenium represent decisive parameters controlling the immune response and regulation of SEPS expression in vivo. Male and female mice fed a selenium-poor diet were supplemented or not with selenite for 3 days and injected with saline or LPS (lipopolysaccharide) 24 h before analysis. Selenium supplementation mitigated the LPS-induced rise in circulating cytokines in male mice. Serum SepP and selenium concentrations decreased in response to LPS, whereas hepatic SepS was specifically up-regulated despite declining selenium concentrations in the liver. Hepatic SepS induction was mainly controlled by post-transcriptional mechanisms and attributed to hepatocytes by analysing transgenic mice. Notably, selenium supplementation was essential for an optimal SepS induction. We conclude that selenoprotein biosynthesis becomes redirected in hepatocytes during the acute-phase response at the expense of dispensable selenoproteins (e.g. SepP) and in favour of SepS expression, thereby causing declining serum selenium and improving liver function. The selenium status and sex control SepS expression and modify cytokine response patterns in serum, which might explain contradictory results on associations of SEPS genotype and inflammatory diseases in clinical studies.
Abstract: Rimexolone is a lipophilic glucocorticoid drug used for local application. Only few data are available describing its effects on immune cell functions. In this study we investigated the effects of rimexolone on the proliferation of human CD4+ T-cells using dexamethasone as standard reference. Isolated CD4+ T-cells were pre-incubated with rimexolone or dexamethasone at different concentrations for 10Â min (10-11/10-8/10-5Â M) and stimulated with anti-CD3/anti-CD28 for 96Â h. Proliferation was determined by flow cytometry. The percentage of dividing cells was significantly reduced by 10-5Â M rimexolone and dexamethasone; however, the average number of cell divisions was unchanged. In addition, production of IL-2 and other cytokines was reduced by both glucocorticoids at 10-5Â M. Interestingly, we observed a rimexolone-induced down-regulation of CD4 expression in unstimulated and non-dividing cells. The inhibitory effects on proliferation and CD4 expression could be blocked by the glucocorticoid-antagonist RU486 and were not due to glucocorticoid-induced apoptosis. Rimexolone and dexamethasone showed a similar potential to induce I[kappa]B[alpha] gene expression. We demonstrate rimexolone and dexamethasone to impair T-cell signalling pathways by rapid non-genomic suppression of the phosphorylation of Akt, p38 and ERK. We conclude that rimexolone and dexamethasone inhibit T-cell proliferation as well as cytokine production of activated CD4+ T-cells in a similar manner. As these inhibitory effects predominantly occur at high concentrations, a relatively high occupation-rate of cytosolic glucocorticoid receptors is needed, but receptor-mediated non-genomic effects may also be involved. It is implied that these effects contribute to the well-known beneficial anti-inflammatory and immunomodulatory effects of glucocorticoid therapy.
Abstract: Human embryonic stem (ES) cells possess an enormous potential for applications in regenerative medicine. However, these cells have several inevitable hurdles limiting their clinical applications, such as transplant rejection and embryo destruction. A milestone recently achieved was the derivation of induced pluripotent stem (iPS) cells by over-expressing combinations of defined transcription factors, namely, OCT4, SOX2, NANOG, and LIN28 or OCT4, SOX2, KLF4, and c-MYC. Human iPS cells exhibit many characteristics identical to those of inner cell mass-derived ES cells. Here, we summarize the generation of human fibroblast-derived iPS cells and discuss the promises and limitations of their use. In addition, by utilising numerous published transcriptome datasets related to ES cells, fibroblast-derived iPS cells, partially induced pluripotent stem cells (PiPSC) and wild type fibroblasts, we reveal similarities (self-renewal signature) and differences (donor cell-type and PiPSC signatures) in genes and associated signaling pathways operative in the induction of pluripotency in fibroblasts. In particular, we highlight that induction of ground state pluripotency is also favoured by the inhibition of epithelial mesenchymal transition (EMT) and hence the induction of mesenchymal epithelial transition (MET). We anticipate that these findings might aid in the establishment of more efficient protocols for inducing pluripotency in somatic cells.
Abstract: Recently it has become clear that the neuronal compartment plays a more important role than previously thought in the pathology of multiple sclerosis. Apart from demyelination, neuronal pathology is apparently largely responsible for the brain atrophy that can be observed early on and throughout the course of the disease. The loss of axons and their neurons in the course of chronic neuroinflammation is a major factor determining long-term disability in patients. The actual steps leading from immune attack against the myelin sheath to neuronal damage are not yet fully clear. Here we review key findings about direct axonal damage processes, demyelination-related neuronal pathology and cell-body pathology, the major pathologic correlates that underlie brain atrophy in MS.
Abstract: New degenerative chain transfer agents, namely 4-(trimethylsilyl)benzyl 4'-(trimethylsilyl)butane-dithioate, 4-(trimethylsilyl)benzyl 3'-(trimethylsilyl)propyl trithiocarbonate and their 3-(trimethylsilyl)benzyl isomers, that are two-fold labeled with complementary trimethylsilyl (TMS) markers, were designed and shown to be powerful tools for universal polymer analysis by conventional (1)H NMR spectroscopy. Their use in controlled free radical polymerization, here the reversible addition-fragmentation chain transfer (RAFT) method, resulted in polymers with low polydispersities up to high molar masses, as well as with defined complementary TMS end groups. Thus, routine (1)H NMR spectra allowed facile determination of the molar masses of polymers of various chemical structures up to at least 10(5) g/mol, and simultaneously provided crucial information about the content of end groups that is typically >95% when polymerizations are correctly performed. Polymerizations were carried out in various solvents for two standard monomers, namely n-butyl acrylate and styrene, as well as for two specialty monomers, so-called inimers, namely 2-(2-chloropropionyloxy)ethyl acrylate and 2-(2-chloropropionyloxy)ethyl acrylamide. The complementary end group markers revealed marked differences in the suitability of commonly used solvents for RAFT polymerization. The results demonstrate-beyond good polymerization control-that the new RAFT agents are universal, powerful tools for facile polymer analysis by routine (1)H NMR spectroscopy, of their absolute molar masses as well as of the content of end groups. This is crucial information, e.g., for the synthesis of high-quality telechelics and, in particular, of block copolymers, which is difficult to obtain by other methods. Preliminary screening experiments indicate that similar uses can be envisaged for analogous ATRP systems.
Abstract: Background-CCN1 is an evolutionary ancient matricellular protein that modulates biological processes associated with tissue repair. Induction at sites of injury was observed in conditions ranging from skin wounds to cardiac diseases, including ischemic and inflammatory cardiomyopathy. Here, we provide evidence of a novel function of CCN1 as a modulator of immune cell migration. xD;Methods and Results-To understand the role of CCN1 in cardiomyopathies and to evaluate its therapeutic potential, we overexpressed CCN1 using an adenoviral hepatotropic vector in murine experimental autoimmune myocarditis, a model of human inflammatory cardiomyopathy. CCN1 gene transfer significantly reduced cardiac disease score and immune cell infiltration. In vivo tracking of hemagglutinin epitope-tagged CCN1 revealed binding to spleen macrophages but not to cardiomyocytes. Unexpectedly, CCN1 therapy left cardiac chemokine and cytokine expression unchanged but instead strongly inhibited the migration of spleen macrophages and lymphocytes, as evidenced by ex vivo transwell assays. In accordance with the ex vivo data, in vitro preincubation with CCN1 diminished transwell migration of human monocytes and abrogated their chemotactic response to monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha , and stromal cell-derived factor-1 alpha. Further mechanistic studies showed that CCN1-driven modulation of immune cell migration is mimicked in part by cyclic RGD peptides currently in clinical evaluation for cancer therapy. xD;Conclusions-Our proof-of-concept study suggests investigation of CCN1 as a novel, endogenous "parent compound" for chemotaxis modulation and of cyclic RGD peptides as a class of partially CCN1-mimetic drugs with immediate potential for clinical evaluation in cardiac diseases associated with chronic pathogenic inflammation. (Circulation.2010;122:2688-2698.)
Abstract: Most polymers used in clinical applications today are materials that have been developed originally for application areas other than biomedicine. Testing the cell- and tissue-compatibility of novel materials in vitro and in vivo is of key importance for the approval of medical devices and is regulated according to the Council Directive 93/42/EEC of the European communities concerning medical devices. In the standardized testing methods the testing sample is placed in commercially available cell culture plates, which are often made from polystyrene. Thus not only the testing sample itself influences cell behavior but also the culture vessel material. In order to exclude this influence, a new system for cell testing will be presented allowing a more precise and systematic investigation by preparing tailored inserts which are made of the testing material. Inserts prepared from polystyrene, polycarbonate and poly(ether imide) were tested for their cytotoxity and cell adherence. Furthermore a proof of principle concerning the preparation of inserts with a membrane-like surface structure and its surface modification was established. Physicochemical investigations revealed a similar morphology and showed to be very similar to the findings to analogous preparations and modifications of flat-sheet membranes.
Abstract: Background: Xenotropic murine leukemia virus-related virus (XMRV), a novel human retrovirus originally identified in prostate cancer tissues, has recently been associated with chronic fatigue syndrome (CFS), a disabling disease of unknown etiology affecting millions of people worldwide. However, several subsequent studies failed to detect the virus in patients suffering from these illnesses or in healthy subjects. Here we report the results of efforts to detect antibody responses and viral sequences in samples from a cohort of German CFS and relapsing remitting multiple sclerosis (MS) patients with fatigue symptoms. xD;Methodology: Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors. Fatigue severity in MS patients was assessed using the Fatigue Severity Scale (FSS). Validated Gag-and Env-ELISA assays were used to screen sera for XMRV antibodies. PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene. In addition, PBMC cultures were exposed to 22Rv1-derived XMRV to assess infectivity and virus production. xD;Conclusion: None of the screened sera from CFS and MS patients or healthy blood donors tested positive for XMRV specific antibodies and all PBMC (and PBMC plus LNCaP) cultures remained negative for XMRV sequences by nested PCR. These results argue against an association between XMRV infection and CFS and MS in Germany. However, we could confirm that PBMC cultures from healthy donors and from CFS patients can be experimentally infected by XMRV, resulting in the release of low levels of transmittable virus.
Abstract: Mutations in the gene encoding transforming growth factor-beta receptor type II (TGFBR2) have been described in patients with Loeys-Dietz syndrome (LDS), Marfan syndrome type 2 (MFS2) and familial thoracic aortic aneurysms and dissections (TAAD). Here, we present a comprehensive and quantitative analysis of TGFBR2 expression, turnover and TGF-beta-induced Smad and ERK signaling activity for nine mutations identified in patients with LDS, MFS2 and TAAD. The mutations had different effects on protein stability, internalization and signaling. A dominant-negative effect was demonstrated for mutations associated with LDS and MFS2. No mutation showed evidence of an immediate cell-autonomous paradoxical activation of TGF-beta signaling. There were no cell biological differences between mutations described in patients with LDS and MFS2. By contrast, R460C, which has been found in familial TAAD but not in MFS2 or LDS, showed a less-severe dominant-negative effect and retained residual Smad phosphorylation and transcriptional activity. TAAD is characterized primarily by thoracic aortic aneurysms or dissections. By contrast, MFS2 is characterized by numerous skeletal abnormalities, and patients with LDS additionally can display craniofacial and other abnormalities. Therefore, our findings suggest that the balance between defects in Smad and ERK signaling might be an important determinant of phenotypic severity in disorders related to mutations in TGFBR2.
Abstract: Bone regeneration is controlled by a variety of biochemical, biomechanical, cellular, and hormonal mechanisms. In particular, physical properties of the substrate such as stiffness and architecture highly influence the proliferation and differentiation of cells. The aim of this work is to understand the influence of scaffold stiffness and cell seeding densities on the formation of tissue by osteoblast cells within polyether urethane scaffolds containing pores of different sizes. MC3T3-E1 preosteoblast cells were seeded on the scaffold, and the amount of tissue formed within the pores was analyzed for culture times up to 49 days by phase contrast microscopy. The authors show that the kinetics of three-dimensional tissue growth in these scaffolds follows two stages and can be described by a universal growth law. The first stage is dominated by cell-material interactions with cell adherence and differentiation being strongly dependent on the polymer material. After a delay time of a few weeks, cells begin to grow within their own matrix, the delay being strongly dependent on substrate stiffness and seeding protocols. In this later stage of growth, three-dimensional tissue amplification is controlled rather by the pore geometry than the scaffold material properties. This emphasizes how geometric constraints may guide tissue formation in vitro and shows that optimizing scaffold architectures may improve tissue formation independent of the scaffold material used. (C) 2010 American Vacuum Society. [DOI: 10.1116/1.3431524]
Abstract: Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.
Abstract: The increasing spectrum of different cartilage repair strategies requires the introduction of adequate non-destructive methods to analyse their outcome in-vivo, i.e. arthroscopically. The validity of non-destructive quantitative ultrasound biomicroscopy (UBM) was investigated in knee joints of five miniature pigs. After 12 weeks, six 5-mm defects, treated with different cartilage repair approaches, provided tissues with different structural qualities. Healthy articular cartilage from each contralateral unoperated knee joint served as a control. The reflected and backscattered ultrasound signals were processed to estimate the integrated reflection coefficient (IRC) and apparent integrated backscatter (AIB) parameters. The cartilage repair tissues were additionally assessed biomechanically by cyclic indentation, histomorphologically and immunohistochemically. UBM allowed high-resolution visualisation of the structure of the joint surface and subchondral bone plate, as well as determination of the cartilage thickness and demonstrated distinct differences between healthy cartilage and the different repair cartilage tissues with significant higher IRC values and a steeper negative slope of the depth-dependent backscatter amplitude AIB(slope) for healthy cartilage. Multimodal analyses revealed associations between IRC and the indentation stiffness. Furthermore, AIB(slope) and AIB at the cartilage-bone boundary (AIB(dC)) were associated with the quality of the repair matrices and the subchondral bone plate, respectively. This ex-vivo pilot study confirms that UBM can provide detailed imaging of articular cartilage and the subchondral bone interface also in repaired cartilage defects, and furthermore, contributes in certain aspects to a basal functional characterization of various forms of cartilage repair tissues. UBM could be further established to be applied arthroscopically in-vivo.
Abstract: Objective Optical coherence tomography (OCT) has gained increasing attention in multiple sclerosis (MS) research and has been suggested as outcome measure for neuroprotective therapies. However, to date it is not clear whether patterns of retinal nerve fiber layer thickness (RNFLT) loss are different in MS compared to other diseases such as glaucoma and data on RNFLT loss in MS patients with or without optic neuritis (ON/NON) have remained inconsistent or even contradictory.Methods In this large cross-sectional study we analyzed the patterns of axonal loss of retinal ganglion cells in MS eyes (n = 262) with and without history of ON (MS/ON: 73 eyes; MS/NON: 189 eyes) and patients eyes with glaucomatous optic disc atrophy (GA: n = 22; 39 eyes) in comparison to healthy control eyes (HC: n = 406 eyes).Results We found that significant average and quadrant RNFLT loss is detectable by OCT in both MS and GA patients compared to healthy controls (p < 0.01). The age- and gender adjusted average and quadrant RNFLT did not differ significantly between MS and GA patients (p > 0.05). Average (p < 0.0001) and quadrant (p < 0.05) RNFL thinning is significantly more severe in MS/ON versus MS/NON eyes, and the extent of RNFL thinning varies across quadrants in MS/ON eyes with the highest degree of RNFLT loss in the temporal quadrant (p < 0.001).Conclusion RNFLT reduction across all four quadrants in MS patients as a whole as well as in MS/NON eyes argues for a diffuse neurodegenerative process. Superimposed inflammatory attacks to the optic nerve may cause additional axonal damage with a temporal preponderance. Future studies are necessary to further evaluate the capacity of OCT to depict disease specific damage patterns.
Abstract: Bone morphogenetic proteins (BMPs) are key regulators of cell fate decisions during embryogenesis and tissue homeostasis. BMPs signal through a coordinated assembly of two types of transmembrane serine/threonine kinase receptors to induce Smad1/5/8 plus non-Smad pathways, such as MAPK and Akt. The recent discovery of BMP receptor inhibitors opened new avenues to study specific BMP signalling and to delineate this effect from TGF-[beta] and Activin signalling. Here we present comprehensive and quantitative analyses on both canonical and non-Smad mediated BMP signalling under Dorsomorphin (DM) and LDN-193189 (LDN) treatment conditions. We demonstrate for the first time, that both compounds affect not only the Smad but also the non-Smad signalling pathways induced by either BMP2, BMP6 or GDF5. The activation of p38, ERK1/2 and Akt in C2C12 cells was inhibited by DM and LDN. In addition "off-target" effects on all branches of BMP non-Smad signalling are presented. From this we conclude that the inhibition of BMP receptors by DM and more efficiently by LDN-193189 affects all known BMP induced signalling cascades.
Abstract: Acrylonitrile-based polymer systems (PAN) are comprehensively explored as versatile biomaterials having various potential biomedical applications, such as membranes for extra corporal devices or matrixes for guided skin reconstruction. The surface properties (e.g. hydrophilicity or charges) of such materials can be tailored over a wide range by variation of molecular parameters such as different co-monomers or their sequence structure. Some of these materials show interesting biofunctionalities such as capability for selective cell cultivation. So far, the majority of AN-based copolymers, which were investigated in physiological environments, were processed from the solution (e.g. membranes), as these materials are thermo-sensitive and might degrade when heated. xD;In this work we aimed at the synthesis of hydrophobic, melt-processable AN-based copolymers with adjustable elastic properties for preparation of model scaffolds with controlled pore geometry and size. For this purpose a series of copolymers from acrylonitrile and n-butyl acrylate (nBA) was synthesized via free radical copolymerisation technique. The content of nBA in the copolymer varied from 45 wt% to 70 wt%, which was confirmed by (1)H-NMR spectroscopy. The glass transition temperatures (T(g)) of the P(AN-co-nBA) copolymers determined by differential scanning calorimetry (DSC) decreased from 58 degrees C to 20 degrees C with increasing nBA-content, which was in excellent agreement with the prediction of the Gordon-Taylor equation based on the T(g)s of the homopolymers. The Young's modulus obtained in tensile tests was found to decrease significantly with rising nBA-content from 1062 MPa to 1.2 MPa. All copolymers could be successfully processed from the melt with processing temperatures ranging from 50 degrees C to 170 degrees C, whereby thermally induced decomposition was only observed at temperatures higher than 320 degrees C in thermal gravimetric analysis (TGA). Finally, the melt processed P(AN-co-nBA) biomaterials were sterilized with ethylene oxide and tested for cytotoxicity in direct contact tests with L929 cells according to the EN DIN ISO standard 10993-5. All tested samples exhibited non-toxic effects on the functional integrity of the cell membrane and the mitochondrial activity. However, the morphology of the cells on the samples was different from that observed on polystyrene as control, indicating slightly cytotoxic effects according to the evaluation guide of the US Pharmacopeial Convention. Thus, the melt-processable, hydrophobic P(AN-co-nBA) copolymers with adjustable mechanical properties are promising candidates for in vitro investigations of tissue growth kinetics.
Abstract: Oxidative phosphorylation and/or glycolysis provide energy, mainly in the form of ATP, which ensures proper functioning of immune cells such as CD4+ T lymphocytes. However, the main substrates, namely oxygen and glucose, are known to remain for a relatively short time in the inflamed tissue and in other clinical situations where immune cells need to function properly. Therefore, we examined the effect of hypoxia and/or lack of glucose on cellular energy metabolism and on cytokine secretion in stimulated human CD4+ T lymphocytes. Human CD4+ T cells were MACS-isolated using peripheral blood obtained from healthy donors. Stimulated cells were incubated in medium with or without glucose for 6Â h in a sealed chamber which led to cumulative hypoxia. During this incubation period, (i) oxygen saturation was measured continuously using a Clark-type electrode, and (ii) samples were taken at different time points in order to quantify for each the viability of cells, intracellular reactive oxygen species (iROS), ATP levels, glycolytic enzyme activity, mRNA expression of hexokinase-1 and superoxide dismutase-1, and concentrations of several different cytokines. Stimulated CD4+ T cells which were incubated under normoxic conditions served as controls. Under hypoxic conditions, lack of glucose exerted a biphasic effect on cellular oxygen consumption: initially higher but later lower respiration rates were measured when compared to conditions where glucose was available. Lack of glucose strongly increased the number of dead cells and the formation of iROS under normoxia but not under hypoxia. Under both normoxic and hypoxic conditions, intracellular ATP levels remained almost unchanged during the incubation period if glucose was present, but decreased significantly in the absence of glucose, despite the enhanced glycolytic enzyme activity. Measurements of stimulated cytokine production demonstrated (i) that cumulative hypoxia stimulates especially the secretion of IL-1[beta], IL-10 and IL-8, and (ii) that lack of glucose results in lower cytokine concentrations. We demonstrate that CD4+ T cells are highly adaptive in bioenergetic terms which ensure their proper function under extreme conditions of glucose and/or oxygen availability as found under physiological and pathophysiological conditions. Hypoxia seems to facilitate inflammatory reactions and angiogenesis.
Abstract: The processing of polymers for blood contacting devices can have a major influence on surface properties. In this study, we fabricated poly(ether imide) (PEI) membranes and films to investigate the effects of the processing on physicochemical surface properties by atomic force microscopy (AFM), scanning electron microscopy, contact angle as well as zeta potential measurements. A static platelet adhesion test was performed to analyze the thrombogenicity of both devices. While contact angle measurements showed similar levels of hydrophobicity and zeta potential values were equivalent, mean surface roughness as well as surface energies in the dispersive part were found to be increased for the PEI membrane. The static platelet adhesion test showed a significantly decreased number of adherent platelets per surface area on the PEI film (178.98 +/- 102.70/45000 mu m(2)) compared to the PEI membrane (504 +/- 314.27/45000 mu m(2)) and, consequently, revealed evidence for higher thrombogenicity of the PEI membrane. This study shows that processing can have a significant effect on platelet adhesion to biomaterials, even though, molar weight was identical. Thrombogenicity of polymer-based cardiovascular devices, therefore, have to be evaluated at the final product level, following the entire processing procedure.
Abstract: Daily nutrition varies considerably among individuals. The number of vegetarians is increasing continuously due to ethical, environmental, religious or other reasons. There is growing concern over their nutritional status with respect to micronutrient deficiencies. Among the essential trace elements, Se is of prime importance as it is part of the active site in selenoproteins. European soil and plants are relatively poor sources of Se, while farm animals are generally supplemented with Se in order to improve their health and avoid deficiency syndromes. We therefore wondered whether German vegetarians display a measurable Se deficiency. To this end, we compared young vegetarians (n 54) and omnivores (n 53). We assessed their Se status by measuring extracellular glutathione peroxidase 3 (GPX3) activity, and concentrations of total serum Se and circulating Se-transport protein selenoprotein P (SEPP). GPX3 activities were not different between the groups, whereas both total Se and SEPP concentrations were reduced to 79.5 and 71.2% in vegetarians compared with omnivores. When splitting the group of vegetarians into vegans (n 26) and vegetarians consuming egg and milk products (n 28), analyses of the Se-dependent biomarkers did not reveal significant differences. We conclude that low serum Se is mirrored by circulating SEPP concentrations, but not by GPX3 activities in marginally supplied individuals. The specific dietary Se sources, divergent metabolic routes of selenomethionine v. selenocysteine and the different saturation kinetics of GPX3 and SEPP probably underlie our contradictory findings. Whether German vegetarians and vegans need to be considered as a Se-deficient group depends on the biomarker chosen.
Abstract: Th1 cells are prominent in inflamed tissue, survive conventional immunosuppression, and are believed to play a pivotal role in driving chronic inflammation. Here, we identify homeobox only protein (Hopx) as a critical and selective regulator of the survival of Th1 effector/memory cells, both in vitro and in vivo. Expression of Hopx is induced by T-bet and increases upon repeated antigenic restimulation of Th1 cells. Accordingly, the expression of Hopx is low in peripheral, naive Th cells, but highly up-regulated in terminally differentiated effector/memory Th1 cells of healthy human donors. In murine Th1 cells, Hopx regulates the expression of genes involved in regulation of apoptosis and survival and makes them refractory to Fas-induced apoptosis. In vivo, adoptively transferred Hopx-deficient murine Th1 cells do not persist. Consequently, they cannot induce chronic inflammation in murine models of transfer-induced colitis and arthritis, demonstrating a key role of Hopx for Th1-mediated immunopathology.
Abstract: Osteoporose ist eine Skeletterkrankung, die nur teilweise durch eine Veränderung der Knochenmineraldichte gekennzeichnet ist. Knochenqualität wird durch eine Vielzahl von kompositionellen und ultrastrukturellen Parametern der mineralisierten Knochenmatrix bestimmt. Im Gegensatz zu radiografischen Methoden tragen Ultraschallwellen durch ihre elastische Wechselwirkung mit dem Knochengewebe Informationen über dessen elastische und ultrastrukturelle Eigenschaften. Quantitative Ultraschallmethoden (QUS) sind erstklassige Alternativen zur radiologischen Abschätzung des Frakturrisikos. Neue Methoden messen direkt an besonders frakturgefährdeten anatomischen Regionen, wie. z.B. dem distalen Radius und dem proximalen Femur. Experimentell erlauben Schallfrequenzen bis in den GHz-Bereich „elastische Einblicke“ bis in die lamelläre Knochenstruktur. Das Potenzial der Kombination hochaufgelöster mikroelastischer Verteilungsmessungen mit numerischen Schallausbreitungssimulationen zur Optimierung neuer QUS-Methoden wird im Folgenden vorgestellt.
Abstract: Th1 and Th17 cells are distinct lineages of effector/memory cells, imprinted for re-expression of IFN-γ and IL-17, by upregulated expression of T-bet and retinoic acid-related orphan receptor γt (RORγt), respectively. Apparently, Th1 and Th17 cells share tasks in the control of inflammatory immune responses. Th cells coexpressing IFN-γ and IL-17 have been observed in vivo, but it remained elusive, how these cells had been generated and whether they represent a distinct lineage of Th differentiation. It has been shown that ex vivo isolated Th1 and Th17 cells are not interconvertable by TGF-β/IL-6 and IL-12, respectively. Here, we show that ex vivo isolated Th17 cells can be converted into Th1/Th17 cells by combined IFN-γ and IL-12 signaling. IFN-γ is required to upregulate expression of the IL-12Rβ2 chain, and IL-12 for Th1 polarization. These Th1/Th17 cells stably coexpress RORγt and T-bet at the single-cell level. Our results suggest a molecular pathway for the generation of Th1/Th17 cells in vivo, which combine the pro-inflammatory potential of Th1 and Th17 cells.
Abstract: BACKGROUND: A 23 year-old female presented to a neurology department with a 3 year history of recurrent episodes involving hearing loss, encephalopathy, focal neurological deficits, and visual field deficits. In the 3 years before presentation, the patient had been treated with methylprednisolone for suspected acute demyelinating encephalomyelitis and peripheral otogenic dysfunction from which she made a complete recovery, and for a visual defect in both eyes caused by bilateral branch retinal arterial occlusion, from which she partially improved and commenced long-term treatment with acetylsalicylic acid. INVESTIGATIONS: Detailed history, clinical examination, extensive laboratory work-up, cerebrospinal fluid analysis, cerebral and spinal MRI, periventricular single-voxel (1)H magnetic resonance spectroscopy, retinal fluorescence angiography, optical coherence tomography, audiometry, neurophysiological work-up (EEG, evoked potentials). DIAGNOSIS: Susac syndrome, characterized by a combination of encephalopathy, branch retinal artery occlusions, and hearing loss. MANAGEMENT: Long-term immunosuppressive treatment with azathioprine (150 mg/day) and prednisolone (10 mg/day), and inhibition of thrombocyte function with acetylsalicylic acid (100 mg/day).
Abstract: Various therapeutic strategies that aim to influence clinical outcome after severe skeletal muscle trauma have been considered. One such method, the local transplantation of stem cells, has been shown to improve tissue regeneration. The number of cells required for successful regeneration, however, remains unclear. The aim of this study was therefore to examine the correlation between the number of transplanted bone marrow-derived mesenchymal stem cells (MSCs) and the resulting muscle function. One week after inducing an open crush trauma in 34 female Sprague Dawley rats, increasing quantities of autologous MSCs (0.1 x 10(6), 1 x 10(6), 2.5 x 10(6), and 10 x 10(6) cells) or saline solution (control group) were transplanted into the left soleus muscle of the rat hind limb. At 4 weeks posttrauma, the outcome was assessed by measuring muscle contraction forces following an indirect fast twitch and tetanic stimulation. A logarithmic dose-response relationship was observed for both maximum twitch and tetanic contraction forces (R(2) = 0.9 for fast twitch [p = 0.004]; R(2) = 0.87 [p = 0.002] for tetanic contraction). The transplantation of 10 x 10(6) cells resulted in the most pronounced improvement of muscle force. MSC therapy represents a promising new tool for the treatment of skeletal muscle trauma that shows potential for aiding in the prevention of severe functional deficiencies. The logarithmic dose-response relationship demonstrates the association between the number of transplanted cells and the resulting muscle forces, as well as the amount of MSCs required for promoting muscular regeneration.
Abstract: Bone Morphogenetic Proteins (BMPs) play an important role during organ development and during regeneration after tissue damage. BMPs signal via transmembrane serine/threonine kinase receptors. From our current understanding heteromeric complexes of type I and type II receptors are required for signal propagation. Presently, three type I and three type I receptors are known to bind BMPs with different affinities. Ligands and receptors eventually oligomerize via defined modes into signaling complexes. Co-receptors recruit into these complexes to either inhibit or to promote signaling. The Smad pathway, initiated by phosphorylation through the activated type I receptors, results in transcriptional regulation of early target genes. However, on its way to the nucleus, Smads represent signaling platforms for other pathways, which eventually finetune BMP signal transduction. We also describe BMP-induced signaling cascades leading to cytoskeletal rearrangements, non-transcriptional and non-Smad pathways. BMPs induce a plethora of different cellular effects ranging from stem cell maintenance, migration, differentiation, proliferation to apoptosis. The molecular mechanism, by which the same ligand induces these manifold effects, depends on the cellular context. Here we try to give a current Picture of the most important players in regulating and directing BMP signaling towards the desired cellular outcome. Examples of BMP action during development, but also physiological and pathophysiological conditions in the adult organism are presented. (C) 2009 Elsevier Ltd. All rights reserved.
Abstract: Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K, T) in patients with multiple synostosis syndrome (SYM1) in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.
Abstract: Cytotoxic T lymphocytes (CTL) control the replication of human cytomegalovirus (CMV). Previous studies assessed the clonotypic composition of CTL specific for individual immunodominant peptides within a certain HLA context. Such an approach has inherent limitations and may not assess the true clonal CTL response in vivo. Here, the clonotypic composition of CMV-specific CTL was determined in HLA-A2, CMV-seropositive kidney transplant recipients and healthy blood donors after stimulation of peripheral blood mononuclear cells with either a pp65 whole-peptide pool or a single immunodominant peptide. Even after stimulation with the whole peptide pool, CMV-specific CTL remained monoclonal or oligoclonal. Regarding intraindividual variation, the CDR3 motifs of the dominant clones were identical to those observed in CTL generated by the single immunodominant peptide. Sequencing of the CDR3 regions demonstrated significant interindividual variation; however, structural homology was observed for immunodominant clonotypes in three individuals. In conclusion, the highly focused T cell receptor repertoire found after stimulation with either a single immunodominant peptide or a peptide pool demonstrates a pivotal role for immunodominant epitopes in the generation of a clonal repertoire. These results provide new insights into the regulation of CMV clonal dominance and may contribute to the design and monitoring of adoptive immunotherapy.
Abstract: Background. BKV reactivation plays the causative role in the development of BKV-associated nephropathy (BKVAN). Because of the lack of effective therapy, early diagnosis of BKV reactivation is paramount for the prevention of BKVAN. Resting in uroepithelial cells, BKV is excreted first in urine before it call be detected in plasma. The present study analyzed predictive value of BK viruria for the development of BK viremia and its possible advantage for the early BKVAN prediction. xD;Methods. Total of 4128 urine and serum samples obtained from renal transplant patients were analyzed for BKV positivity by real-time polymerase chain reaction in 433 patients in cross-sectional and in 233 patients in longitudinal manner, respectively. The prospective longitudinal analysis included seven measurements during the first posttransplant year. xD;Results. A total of 7% and 19% patients were positive for BKV in serum and Urine, respectively. Sustained BK viruria showed sensitivity of 100% and specificity of 94% for BK viremia and was associated with significantly higher level of BK load than the patients with transient viruria (P<0.01). Interestingly, BK viremia was preceded by BK viruria: the peak of viral load and number of positive patients appeared during the third and fifth posttransplant month for urine and serum, respectively. BKVAN diagnosed in 21.4% of patient with persistent BK viruria appeared 5 and 11 weeks after BKV reactivation in serum and urine, respectively, was detected. xD;Conclusion. Sustained BK viruria is a reliable marker allowing all early identification of patients at high risk of BKVAN development and therefore assure precocious therapeutic interventions.
Abstract: Integration of multiple signals into the canonical BMP/Smad pathway poses a big challenge during the course of embryogenesis and tissue homeostasis. Here, we show that cyclic guanosine 3′,5′-monophosphate (cGMP)-dependent kinase I (cGKI) modulates BMP receptors and Smads, providing a novel mechanism enhancing BMP signalling. cGKI, a key mediator of vasodilation and hypertension diseases, interacts with and phosphorylates the BMP type II receptor (BMPRII). In response to BMP-2, cGKI then dissociates from the receptors, associates with activated Smads, and undergoes nuclear translocation. In the nucleus, cGKI binds with Smad1 and the general transcription factor TFII-I to promoters of BMP target genes such as Id1 to enhance transcriptional activation. Accordingly, cGKI has a dual function in BMP signalling: (1) it modulates BMP receptor/Smad activity at the plasma membrane and (2) after redistribution to the nucleus, it further regulates transcription as a nuclear co-factor for Smads. Consequently, cellular defects caused by mutations in BMPRII, found in pulmonary arterial hypertension patients, were compensated through cGKI, supporting the positive action of cGKI on BMP-induced Smad signalling downstream of the receptors.
Abstract: In the course of autoimmune CNS inflammation, inflammatory infiltrates form characteristic perivascular lymphocyte cuffs by mechanisms that are not yet well understood. Here, intravital two-photon imaging of the brain in anesthetized mice, with experimental autoimmune encephalomyelitis, revealed the highly dynamic nature of perivascular immune cells, refuting suggestions that vessel cuffs are the result of limited lymphocyte motility in the CNS. On the contrary, vessel-associated lymphocyte motility is an actively promoted mechanism which can be blocked by CXCR4 antagonism. In vivo interference with CXCR4 in experimental autoimmune encephalomyelitis disrupted dynamic vessel cuffs and resulted in tissue-invasive migration. CXCR4-mediated perivascular lymphocyte movement along CNS vessels was a key feature of CD4+ T cell subsets in contrast to random motility of CD8+ T cells, indicating a dominant role of the perivascular area primarily for CD4+ T cells. Our results visualize dynamic T cell motility in the CNS and demonstrate differential CXCR4-mediated compartmentalization of CD4+ T-cell motility within the healthy and diseased CNS.
Abstract: Myostatin, a negative regulator of muscle growth, has recently been found to be expressed in tendons. Myostatin-deficient mice have weak and brittle tendons, which suggest that myostatin could be important for tendon maintenance. Follistatin expression in the callus tissue after tendon transection is influenced by loading. We found that follistatin antagonises myostatin, but not GDF-5 or OP-1 in vitro. To study if myostatin might play a physiological role in soft tissue, we transected 64 rat Achilles tendons and studied the gene expression for myostatin and its receptors at four different time-points during healing. Intact tendons were also studied. All samples were studied with or without mechanical loading. Unloading was achieved with botulinum toxin injections in the calf muscles. The expression of the myostatin gene was more than 40 times higher in intact tendons than in the callus tissue during tendon healing. The expression of myostatin was also influenced by loading status in both intact and healing tendons. Thereafter, we measured the mechanical properties of healing tendons after local myostatin administration. This treatment increased the volume and the contraction of the callus after 8 days, but did not improve its strength. Our results indicate that myostatin plays a positive role in tendon maintenance and that exogenous protein administration stimulates proliferation and growth of early repair tissue. However, no effect on further development towards connective tissue formation was found. xD; xD; xD;Read More: http://informahealthcare.com/doi/abs/10.1080/08977190903052539
Abstract: We addressed the effect of post-transplant lymphoproliferative disorder (PTLD) treatment with rituximab monotherapy or CHOP-based chemotherapy (± rituximab) after upfront immunosuppression reduction (IR) on renal graft function in a longitudinal analysis of 58 renal transplant recipients with PTLD and 610 renal transplant controls. Changes in the estimated glomerular filtration rate over time were calculated from a total of 6933 creatinine measurements over a period of >1 year using a linear mixed model with random and fixed effects. Renal graft function significantly improved with treatment of PTLD, especially in the chemotherapy subgroup. Patients treated with IR+chemotherapy ± rituximab had a noninferior graft function compared with untreated controls suggesting that the negative impact of IR on the renal graft function can be fully compensated by the immunosuppressive effect of CHOP. The immunosuppressive effect of single agent rituximab may partially compensate the negative impact of IR on the graft function. Thus, it is possible to reduce immunosuppression when using chemotherapy to treat PTLD.
Abstract: LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies, but their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. Microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells revealed that a subset of NF-[kappa]B target genes, including IL-6 and IL-8, are upregulated in LMX1B-expressing cells. Inhibition of NF-[kappa]B activity by short interfering RNA-mediated knock-down of p65 impairs, while activation of NF-[kappa]B activity by TNF-[alpha] synergizes induction of NF-[kappa]B target genes by LMX1B. Chromatin immunoprecipitation demonstrated that LMX1B binds to the proximal promoter of IL-6 and IL-8 in vivo, in the vicinity of the characterized [kappa]B site, and that LMX1B recruitment correlates with increased NF-[kappa]B DNA association. IL-6 promoter-reporter assays showed that the [kappa]B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of NF-[kappa]B target genes is affected in the kidney of Lmx1b-/- knock-out mice, thus supporting the biological relevance of our findings. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-[kappa]B target genes in cooperation with nuclear p50/p65 NF-[kappa]B.
Abstract: 10.1002/stem.49.abs Progenitor cells such as mesenchymal stem cells (MSCs) have elicited great hopes for therapeutic augmentation of physiological regeneration processes, e.g., for bone fracture healing. However, regeneration potential decreases with age, which raises questions about the efficiency of autologous approaches in elderly patients. To elucidate the mechanisms and cellular consequences of aging, the functional and proteomic changes in MSCs derived from young and old Sprague–Dawley rats were studied concurrently. We demonstrate not only that MSC concentration in bone marrow declines with age but also that their function is altered, especially their migratory capacity and susceptibility toward senescence. High-resolution two-dimensional electrophoresis of the MSC proteome, under conditions of in vitro self-renewal as well as osteogenic stimulation, identified several age-dependent proteins, including members of the calponin protein family as well as galectin-3. Functional annotation clustering revealed that age-affected molecular functions are associated with cytoskeleton organization and antioxidant defense. These proteome screening results are supported by lower actin turnover and diminished antioxidant power in aged MSCs, respectively. Thus, we postulate two main reasons for the compromised cellular function of aged MSCs: (a) declined responsiveness to biological and mechanical signals due to a less dynamic actin cytoskeleton and (b) increased oxidative stress exposure favoring macromolecular damage and senescence. These results, along with the observed similar differentiation potentials, imply that MSC-based therapeutic approaches for the elderly should focus on attracting the cells to the site of injury and oxidative stress protection, rather than merely stimulating differentiation. STEM CELLS 2009;27:1288–1297
Abstract: Irreversible or plastic deformation in bone is associated with both permanent plastic strain as well as localized microdamage. Whereas mechanisms at the molecular and mesoscopic level have been proposed to explain aspects of irreversible deformation, it quantitative correlation of mechanical yielding, microstructural deformation, and macroscopic plastic strain does not exist. To address this issue, we developed and applied a two-dimensional image correlation technique to the tensile deformation of bovine fibrolamellar bone, to determine the spatial distribution of strain fields at the length scale of 10 mu m to 1 mm in bone during irreversible tensile deformation. We find that tensile deformation is relatively homogeneous in the elastic regime and starts at the yield point., showing regions of locally higher strain. Multiple regions of high deformation can exist at the same time over a length scale of 1 to 10 mm. Macroscopic fracture always Occurs at one of the locally highly deformed regions, but the selection of which region cannot be predicted. Locally, strain rates call be enhanced by a factor of 3 to 10 over global strain rates in the highly deformed zones and are lower but always positive in all other regions. Light microscopic imaging shows the onset of structural "banding" in the regions of deformation, which is most likely correlated to microstructural damage at the inter- and intrafibrillar level.
Abstract: Background: Overproduction of pro-inflammatory cytokines is a well established factor in the progression of chronic heart failure (CHF). Changes in cellular immunity have not been widely studied, and the impact of standard medication is uncertain. Here we investigate whether a leukocyte redistribution occurs in CHF and whether this effect is influenced by beta-blocker therapy. xD;Methodology: We prospectively studied 75 patients with systolic CHF (age: 68 +/- 11 years, left ventricular ejection fraction 32 +/- 11%, New York Heart Association class 2.5 +/- 0.7) and 20 age-matched healthy control subjects ( age: 63 +/- 10 years). We measured the response of cells to endotoxin exposure in vitro, analysed subsets of lymphocytes using flow cytometry, and assessed plasma levels of the pro-inflammatory markers interleukin 1, 6, tumor necrosis factor-alpha, and soluble tumor necrosis factor receptors 1 and 2. xD;Principal findings: While no differences in the number of leukocytes were noted between patients with CHF and healthy controls, we detected relative lymphopenia in patients with CHF (p < 0.001 vs. control), mostly driven by reductions in T helper cells and B cells (both p < 0.05). The number of neutrophils was increased (p < 0.01). These effects were pronounced in patients who were beta-blocker naive (32% of all patients with CHF). Increased plasma levels of soluble tumor necrosis receptor-1 correlated with the relative number of lymphocyte subsets. xD;Conclusions: In patients with CHF, we detected a redistribution of leukocyte subsets, i.e. an increase in neutrophils with relative lymphopenia. These effects were pronounced in patients who were beta-blocker naive. The underlying mechanism remains to be elucidated.
Abstract: Objective. Inflamed tissues are usually characterized by low oxygen levels. We investigated whether pathophysiological hypoxia (pO(2) < 1%) as found in the rheumatoid synovium Modulates the transcriptome of human CD4+ T cells. xD;Methods. We analyzed the extent to which hypoxia influences the transcriptome in the rheumatoid synovium according to a gene Cluster reflecting adaptation to low oxygen levels. Hypoxia-inducible factor-1 alpha (HIF-1 alpha) was detected in the rheumatoid synovium using immunohistochemistry. Isolated human CD4+ T cells were exposed to hypoxia and analyzed using microarray analysis, quantitative polymerase chain reaction, and immunoblot detection. xD;Results. In rheumatoid arthritis (RA) synovial tissue samples, hypoxia modulates the transcription profile. This profile is similar, but not identical, to that found in isolated CD4+ T cells incubated under hypoxic conditions. We show that HIF-1 alpha is expressed in synovial tissue samples and in hypoxic CD4+ cells; and that hypoxia directly affects differential gene expression in human T cells with up to 4.8% modulation of the transcriptome. Functional genome analysis revealed substantial effects of hypoxia oil immune response, transcriptional regulation, protein modification, cell growth and proliferation, and cell metabolism. xD;Conclusion. Severe hypoxia, a feature of joint inflammation, considerably modulates the transcriptome of cells found in the rheumatoid synovium. Human CD4+ T cells adapt to hypoxic conditions mainly by HIF-1-driven effects oil the transcriptome reflecting a profound influence on immune functions. Thus, hypoxia must be taken into account when therapeutically targeting inflammation. (First Release Nov 1 2009; J Rheumatol 2009:36:2655-69; doi:10.3899/jrheum.090255)
Abstract: Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply: hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were Performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm H g [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This Study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions Were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This Study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, Pressure and temperature. (c) 2008 Elsevier Inc. All rights reserved.
Abstract: Autologous mesenchymal stem cells (MSCs) have been shown to improve the functional outcome after skeletal muscle trauma. The mechanisms behind this improvement have to be answered prior to a future clinical application. We investigated for the first time the in vivo distribution and behavior of MSCs after local transplantation into a severely injured muscle with magnetic resonance imaging (MRI). Autologous rat MSCs were labeled with very small iron oxide nanoparticles (VSOPs) and transplanted into the soleus muscle 1 week after an open crush injury. Distribution and migration of the cells were evaluated in vivo over time by the repeated performance of high-resolution MRI at 7T. Three and 6 weeks after transplantation, the muscles were histologically analyzed. The labeled MSCs could be visualized inside the traumatized muscles 24 h after transplantation showing characteristic signal reductions in T-2*-weighted sequences. The hypointense signal could be followed over 6 weeks and could be easily discriminated from the structures of the injured muscle. The cell pools did not migrate inside the muscle and showed a decrease in volume over time. Prussian blue-stained histologic sections showed a topographical correlation of the respective MRI signal and nanoparticle-labeled cells. Fusion events of marked cells with regenerating myofibers could be observed. The presented study demonstrates for the first time the feasibility of an in vivo tracking of MSCs with MRI after a severe skeletal muscle injury. The investigated method can be a powerful tool both in experimental setups and in possible clinical applications of stem cell-supported skeletal muscle regeneration.
Abstract: The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor kappa B (NF-kappa B)-dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-kappa B, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-gamma, IL-2, and tumor necrosis factor-alpha, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.
Abstract: The molecular mechanisms for plastic deformation of bone tissue are not well understood. We analysed temperature and strain-rate dependence of the tensile deformation behaviour in fibrolamellar bone, using a technique originally developed for studying plastic deformation in metals. We show that, beyond the elastic regime, bone is highly strain-rate sensitive, with an activation volume of ca 0.6 nm3. We find an activation energy of 1.1 eV associated with the basic step involved in the plastic deformation of bone at the molecular level. This is much higher than the energy of hydrogen bonds, but it is lower than the energy required for breaking covalent bonds inside the collagen fibrils. Based on the magnitude of these quantities, we speculate that disruption of electrostatic bonds between polyelectrolyte molecules in the extrafibrillar matrix of bone, perhaps mediated by polyvalent ions such as calcium, may be the rate-limiting elementary step in bone plasticity.
